Chromatography membranes formed by thiol-ene or thiol-yne click polymerization reactions

ABSTRACT

Disclosed are composite materials and methods of making them. The composite materials comprise a support member and a cross-linked gel, wherein the cross-linked gel is a polymer synthesized by thiol-ene or thiol-yne polymerization and cross-linking. The cross-linked gel may be functionalized by a thiol-ene or thiol-yne grafting reaction, either simultaneously with the polymerization or as the second step in a two-step procedure. The composite materials are useful as chromatographic separation media.

RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalPatent Application Ser. No. 62/118,577, filed Feb. 20, 2015, thecontents of which are hereby incorporated by reference.

BACKGROUND

“Click chemistry” is the class of reactions that resemble natural,biochemical reactions, with the following attributes: highly efficient,“spring-loaded” reactions that proceed rapidly to high yield; highlyselective reactions that produce no (or few) side products and aretolerant of multiple functional groups; and reactions that proceed undermild reaction conditions, such as at low temperatures (e.g., ambient) orin aqueous solutions.

Click chemistry has grown to encompass a range of chemical reactions,such as Diels-Alder reactions, copper-catalyzed alkene-azidecycloaddition (CuAAC), thiol-maleimide addition reactions, andthiol-alkene and thiol-alkyne addition reactions.

The term “thiol-ene” is generally used to describe the hydrothiolationaddition of a thiol to any of a wide variety of unsaturated functionalgroups, such as maleimides, acrylates, and norbornenes, in addition tounactivated carbon-carbon double bonds. In some cases, the reaction cantake place not only via the classical radical addition mechanism, butalso with Michael-type nucleophilic addition. The term “thiol-yne” isused to describe counterpart hydrothiolation methods using an alkyne inplace of an alkene. In general, the thiol-ene and thiol-yne reactionsare conducted under photo-initiated radical conditions and proceed via atypical chain growth process with initiation, propagation, andtermination steps.

The thiol-ene and -yne click reactions have many attractive features forpolymer synthesis. The reactions are rapid, stereo-specific, insensitiveto water, and can provide a variety of polymer functionalities throughthe use of various thiol and/or alkene/alkyne functionalized monomers.By using di-, tri-, and tetra-functionalized thiol and alkene/alkynemonomers, it is possible to perform thiol-ene and -yne click reactionsto build new materials with a variety of chemical functionalities. Thesereactions may also result in more highly organized polymeric networks,in comparison to similar acrylate polymers.

There exists a need for separation or chromatography media that can beeasily made by fast, efficient, and easily-controllable polymerizationreactions, and easily modified. These media must also display highselectivity and high flow velocity, low back pressure, be inexpensive,and allow for long column-lifetimes, short process-times, and overalloperational flexibility.

SUMMARY

In certain embodiments, the invention relates to a composite material,comprising:

a support member, comprising a plurality of pores extending through thesupport member; and

a cross-linked gel, wherein the cross-linked gel comprises a polymerderived from a first monomer and a first cross-linker;

wherein

the cross-linked gel is located in the pores of the support member;

the first monomer comprises two thiol functional groups; and

the first cross-linker comprises (i) at least three carbon-carbon doublebonds, (ii) at least two carbon-carbon triple bonds, or (iii) at leastone carbon-carbon triple bond and at least one carbon-carbon doublebond.

In certain embodiments, the invention relates to a composite material,comprising:

a support member, comprising a plurality of pores extending through thesupport member; and

a cross-linked gel, wherein the cross-linked gel comprises a polymerderived from a first monomer, a second monomer, and a firstcross-linker;

wherein

the cross-linked gel is located in the pores of the support member;

the first monomer comprises two thiol functional groups;

the second monomer comprises two carbon-carbon double bonds; and

the first cross-linker comprises (i) at least three thiol functionalgroups, (ii) at least three carbon-carbon double bonds, (iii) at leasttwo carbon-carbon triple bonds, or (iv) at least one carbon-carbontriple bond and at least one carbon-carbon double bond.

In certain embodiments, the invention relates to a method of making acomposite material, comprising the steps of:

combining a first monomer a first cross-linker, a photoinitiator, and asolvent, wherein the first monomer comprises two thiol functionalgroups; and the first cross-linker comprises (i) at least threecarbon-carbon double bonds, (ii) at least two carbon-carbon triplebonds, or (iii) at least one carbon-carbon triple bond and at least onecarbon-carbon double bond, thereby forming a monomeric mixture;

contacting a support member with the monomeric mixture, thereby forminga modified support member; wherein the support member comprises aplurality of pores extending through the support member, and the averagepore diameter of the pores is about 0.1 to about 25 μm;

covering the modified support member with a polymeric sheet, therebyforming a covered support member; and

irradiating the covered support member for a period of time, therebyforming a composite material.

In certain embodiments, the invention relates to a method of making acomposite material, comprising the steps of:

combining a first monomer, a second monomer, a first cross-linker, aphotoinitiator, and a solvent, wherein the first monomer comprises twothiol functional groups; the second monomer comprises two carbon-carbondouble bonds; and the first cross-linker comprises (i) at least threethiol functional groups, (ii) at least three carbon-carbon double bonds,(iii) at least two carbon-carbon triple bonds, or (iv) at least onecarbon-carbon triple bond and at least one carbon-carbon double bond,thereby forming a monomeric mixture;

contacting a support member with the monomeric mixture, thereby forminga modified support member; wherein the support member comprises aplurality of pores extending through the support member, and the averagepore diameter of the pores is about 0.1 to about 25 μm;

covering the modified support member with a polymeric sheet, therebyforming a covered support member; and

irradiating the covered support member for a period of time, therebyforming a composite material.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the monomeric mixture further comprisesa plurality of end-group precursors; and the end-group precursors aremolecules having a thiol functional group or molecules having anunsaturated carbon-carbon bond.

In certain embodiments, the invention relates to any one of theaforementioned methods, further comprising the steps of:

contacting the composite material with a mixture comprising aphotoinitiator and a plurality of end-group precursors, wherein theend-group precursors are molecules having a thiol functional group ormolecules having an unsaturated carbon-carbon bond, thereby forming agrafting mixture; and

irradiating the grafting mixture for a period of time, thereby forming amodified composite material.

In certain embodiments, the invention relates to a method, comprisingthe step of:

contacting at a first flow rate a first fluid comprising a substancewith any of the composite materials described herein, thereby adsorbingor absorbing a portion of the substance onto the composite material.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 tabulates the reaction components (wt. %) and solvents (wt. %)used in the preparation of membranes formed by click alkene reactionsusing TEGDV as a co-monomer.

FIG. 2 tabulates the reaction components (wt. %) and solvents (wt. %)used in the preparation of membranes formed by click alkene reactionswith TEGDV as a co-monomer and varying amounts of initiator.

FIG. 3 tabulates the reaction components (wt. %) and solvents (wt. %)used in the preparation of membranes formed by click alkene reactionswith TEGDV and DATA as co-monomers.

FIG. 4A tabulates the reaction components (wt. %) and solvents (wt. %)used in the preparation of membranes formed by click alkene reactionswith DATA as a co-monomer.

FIG. 4B tabulates the reaction components (wt. %) and solvents (wt. %)used in the preparation of membranes formed by click alkene reactionswith DATA as a co-monomer, at a higher concentration relative to theexperiment that is the subject of FIG. 4A.

FIG. 5A depicts an ESEM image of a composite membrane formulated withDATA as co-monomer formulated with an alkene-to-thiol ratio (calculatedby number of reactive functional groups present, where one alkyne isequivalent to two alkenes because each alkyne may react with two thiols)of 0.96 (EN-149).

FIG. 5B depicts an ESEM image of a composite membrane formulated withDATA as co-monomer formulated with an alkene-to-thiol ratio (calculatedby number of reactive functional groups present, where one alkyne isequivalent to two alkenes because each alkyne may react with two thiols)of 1.05 (EN-151).

FIG. 6 depicts an ESEM image of a composite membrane (EN-124) formulatedwith DATA as co-monomer; the alkene-to-thiol ratio in the polymerizationmixture for making this membrane was 1.27.

FIG. 7 tabulates the reaction components (wt. %) and solvents (wt. %)for membranes formed by click alkene reactions with octadiyne as anadditional crosslinker.

FIG. 8A depicts an ESEM image of a composite membrane prepared withoctadiyne as an additional crosslinker prepared with an alkene-to-thiolratio of 1.074 (EN-134).

FIG. 8B depicts an ESEM image of a composite membrane prepared withoctadiyne as an additional crosslinker prepared with an alkene-to-thiolratio of 1.18 (EN-120).

FIG. 9 tabulates the reaction components (wt. %) and solvents (wt. %)used in the preparation of membranes formed by click alkene reactionsand grafted with carboxylate moieties via a second click reaction afterpolymerization.

FIG. 10 depicts a graphical representation of the water flux of variouscomposite membranes of the invention before being grafted withcarboxylate moieties (left bar) and after being grafted with carboxylatemoieties (second left bar). Upon exposure to pH 5, the flux increases(second right bar). Upon exposure to 0.1 M NaOH, the flux decreasesagain (right bar).

FIG. 11 depicts a graphical representation of the water flux of variouscomposite membranes of the invention before being grafted withcarboxylate moieties (left bar), after being grafted in water withcarboxylate moieties (center bar), and after being grafted in DMAc withcarboxylate moieties (right bar).

FIG. 12 tabulates the binding capacity of three membranes modified withprotein A in the absence of thiol-functionalized additives (A), and inthe presence of thiol-functionalized additives (B and C).

FIG. 13 tabulates the reaction components (wt. %) and solvents (wt. %)used in the preparation of membranes formed by click alkene reactionswith DATA as a co-monomer and a solvent system comprising, for example,triethylene glycol or tetraethylene glycol.

FIG. 14 depicts an SEM image of CLK-EN-298 membrane.

FIG. 15 tabulates the reaction components (wt. %) and solvents (wt. %)used in the preparation of membranes formed by click alkene reactionswith PETM as a co-crosslinker.

FIG. 16 depicts an SEM image of EN-325 membrane made using a tetrathiol(PETM) cross-linker.

FIG. 17 tabulates the reaction components (wt. %) and solvents (wt. %)used in the preparation of membranes formed by click alkene reactionswith octadiyne as a co-crosslinker.

FIG. 18 depicts an SEM image of the CLK-EN-361 membrane.

FIG. 19 tabulates the reaction components (wt. %) and solvents (wt. %)used in the preparation of HIC membranes formed by one-steppolymerization and functionalization.

FIG. 20 depicts a schematic representation of a two-step graft extensionprocess beginning with an alkene-functionalized membrane.

FIG. 21 tabulates the reaction components (wt. %) and solvents (wt. %)used in a double polymerization reaction.

FIG. 22 tabulates the reaction components (mol. %) used in a doublepolymerization reaction, and the properties of the resulting membranes.

DETAILED DESCRIPTION

Overview

While recent work has focused on using thiol-ene and -yne clickchemistry to produce various functionalized polymer networks, primarilyfor film, coating, and dendrimer applications, the approach has not beenapplied to the production of macroporous network polymer membranesuseful for liquid separation processes. In certain embodiments, theinvention relates to the use of thiol-ene and -yne click chemistry forthe generation of cross-linked polymer membranes suitable for liquidchromatography applications. In certain embodiments, the cross-linkedmembranes are further grafted with chemical functional groups ormolecular species. In certain embodiments, generation of thecross-linked membrane by polymerization, and modification of thecross-linked membrane by grafting, are carried out via highly efficientthiol-ene and/or -yne click reactions (in a one- or two-step procedure).

In certain embodiments, thiol-ene and -yne click chemistry is employedto make a cross-linked polymer that is supported by a fibrous substrate,thereby forming a composite membrane. In certain embodiments, thecross-linked polymers in the composite membranes contain residualreactive groups, such as thiols or unsaturated carbon-carbon bonds, thatmay be used to attach various chemical compounds or molecular speciesvia additional click reactions.

In certain embodiments, the cross-linked polymer is macroporous.Porosity within the polymers may be promoted during polymerization bydegree of crosslinking, solvent exclusion of the polymeric chain duringthe formation of the polymer network, or some combination of both.

The degree of crosslinking in the polymer may be tuned by adjusting themonomer ratio. Specifically, the alkene-to-thiol ratio is considered toensure adequate porosity. The chain length of the polymers in thepolymeric network and, therefore, the degree of crosslinking may also becontrolled by using specific monomers that impart specificphysicochemical properties to the final polymer and membrane. These“tuning” monomers can affect the interaction of the polymer chain withthe solvent system. Moreover, the hydrophilicity/hydrophobicity of thesemonomers can affect the final aqueous swelling properties of theresultant gel and the hydrophilic/hydrophobic surface properties of thepolymer network.

Controlling the porosity of the polymer network requires care when usingclick chemistry because the thiol-ene reaction is so fast. As a result,the movement of the growing chain may be restricted from forming pores.To minimize this undesirable result, the solvent system and monomer areselected to ensure an adequate driving force exists to exclude thegrowing polymer chains from solution at a certain point, thereby formingmacropores. Specifically, the mixture of solvents and non-solvents istuned to provide a suitable reaction system that can initially dissolveall of reactants but serves as a poor solvent for the cross-linkedpolymer chains as they grow to be larger than a certain molecularweight. A solvent system with too high a proportion of poor solvent (forthe polymer chains) can lead to a rapid precipitation of growing polymerchains, which decreases porosity.

In general, many highly porous and non-rigid polymeric materials arerelatively weak and are unable to withstand the pressures generatedduring typical membrane separation processes (e.g., liquidchromatography). Therefore, in order to make membranes that aremechanically suitable, in certain embodiments a composite materialcomprising both a porous substrate (such as a woven substrate made ofthe chemically inert polypropylene) and a porous cross-linked polymer isproduced by synthesizing the polymer directly within the substratepores.

In certain embodiments, when examined using environmental scanningelectron microscopy (ESEM), the composite materials showed awell-connected gel network that is incorporated within the substratefibres.

In certain embodiments, the composite materials of the invention can beeffectively used in both “bind-elute” and “flow-through” modes.

“Bind-elute mode” as used herein, refers to an operational approach tochromatography in which the buffer conditions are established so thatboth a target protein and undesired contaminants bind to thechromatographic support or composite material. Fractionation of targetprotein from the other components is achieved subsequently by changingthe conditions such that the target protein and contaminants are elutedseparately. In certain embodiments, the membranes described herein maybe used in “bind-elute mode” featuring high dynamic binding capacitiesat high conductivity, high volume throughput and selectivity. In certainembodiments, the amount of the target protein in the eluent is reducedby about 50% to about 99%. In certain embodiments, the eluent is reducedin aggregates of the target protein by about 90%, about 91%, about 92%,about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, orabout 99%.

As used herein, the term “flow-through mode” refers to an operationalapproach to chromatography in which the buffer conditions areestablished so that the intact target protein flows through the membraneupon application while contaminants are selectively retained. In certainembodiments, the membranes described herein may be used in “flow-throughmode” in a post-protein A purification process to remove keycontaminants, such as DNA, host cell proteins (HCP), leached protein A,undesirable aggregates, and viruses in a single step.

Various Characteristics of Exemplary Composite Materials

Composition of the Gels

In certain embodiment, the cross-linked polymers may be formed by thereaction between a dithiol monomer and a tri-vinyl monomer or alkynemonomer, which serve as crosslinkers. See Scheme 1A and Scheme 1B. Incertain embodiments, additional monomers may be added to tune the finalchemical, physical, and mechanical properties of the polymer. In certainembodiments, the cross-linked polymers may be formed by the reactionbetween a diene monomer and a tri-thiol monomer, which serves as acrosslinker.

In certain embodiments, the cross-linked polymers may be furtherfunctionalized by grafting the cross-linked chains with a graftingmoiety. In certain embodiments, the grafting moiety is a thiol or analkene that also has additional chemical functionalities. In certainembodiments, the cross-linked polymer is formed and grafted in a singlestep (“one pot” approach, Scheme 2). For example, the inclusion ofmercaptosuccininc acid in the polymerization mixture will result in apolymer with carboxylic acid functionality. The inclusion of cysteamine(or alternatively allyl amine) will result in polymer with aminefunctionality. The use of mercaptoethanesulfonic acid (or alternativelysodium allylsulfonate) will incorporate sulfonic acid groups andnegative charges to the polymer network.

In certain embodiments, the cross-linked polymer may be functionalizedby post-polymerization modification. In this two-step method, the excessthiol or alkene groups generated during the thiol-alkene polymerizationare modified during a separate grafting step (Scheme 3). By controllingthe thiol-to-alkene monomer feed ratio, the final polymers can have asurplus of either alkene or thiol groups. Either functional group can beused subsequently in a grafting reaction, such as a click reaction, tofurther modify the final polymer chemistry or functionality. In certainembodiments, this approach is useful in making polymeric membranes thatcontain various ligands useful for chromatographic separation ofbiomolecules (e.g., proteins). For example, this approach can be used tointroduce to the membrane ion exchange functionalities (carboxylate,sulfonate, quaternary ammonium, amine), hydrophobic interaction moieties(such as octyl group by using 1-octanethiol or 1-octene), andbiomolecules for bio-affinity chromatography (such as cysteine-protein Afor monoclonal antibody purification).

In certain embodiments, thiol-ene grafting is an attractive option forattaching biomolecules to the cross-linked polymer of the membrane. Thereaction is fast, can be carried out efficiently in aqueous media, workswell at room temperature, and can be photo-initiated using a relativelylong wavelength light (365 nm), which has very limited effect on proteinbioactivity. In addition, it can allow for controlled biomoleculeattachment, which can be advantageous in terms of preserving bioactivityand 3D structure of the biomolecule.

In certain embodiments, it is possible to immobilize onto the compositematerials described herein any biomolecule that has free thiolfunctionality. This can be very useful in making bio-affinity membranesfor bioseparation or bio-catalysis membranes (by immobilizingenzyme(s)). In certain embodiments, the composite materials may befunctionalized with oligonucleotide probes for DNA detection.

Porous Support Member

In some embodiments, the porous support member contains pores of averagediameter of about 0.1 to about 50 μm.

In some embodiments, the porous support member has a volume porosity ofabout 40% to about 90%.

In certain embodiments, the porous support is flat.

In certain embodiments, the porous support is disk-shaped.

Many porous substrates or membranes can be used as the support member.In some embodiments, the porous support member is made of polymericmaterial. In certain embodiments, the support may be a polyolefin, whichis available at low cost. In certain embodiments, the polyolefin may bepoly(ethylene), poly(propylene), or poly(vinylidene difluoride).Extended polyolefin membranes made by thermally induced phase separation(TIPS), or non-solvent induced phase separation are mentioned. Incertain embodiments, the support member may be made from naturalpolymers, such as cellulose or its derivatives. In certain embodiments,suitable supports include polyethersulfone membranes,poly(tetrafluoroethylene) membranes, nylon membranes, cellulose estermembranes, fiberglass, or filter papers.

In certain embodiments, the porous support is composed of woven ornon-woven fibrous material, for example, a polyolefin, such aspolypropylene. Such fibrous woven or non-woven support members can havepore sizes larger than the TIPS support members, in some instances up toabout 75 μm. The larger pores in the support member permit formation ofcomposite materials having larger macropores in the macroporous gel.Non-polymeric support members can also be used, such as ceramic-basedsupports. The porous support member can take various shapes and sizes.

In some embodiments, the support member is in the form of a membrane.

In some embodiments, the support member has a thickness from about 10 toabout 2000 μm, from about 10 to about 1000 μm, or from about 10 to about500 μm.

In other embodiments, multiple porous support units can be combined, forexample, by stacking. In one embodiment, a stack of porous supportmembranes, for example, from 2 to 10 membranes, can be assembled beforethe gel is formed within the void of the porous support. In anotherembodiment, single support member units are used to form compositematerial membranes, which are then stacked before use.

Relationship Between Gel and Support Member

The gel may be anchored within the support member. The term “anchored”is intended to mean that the gel is held within the pores of the supportmember, but the term is not necessarily restricted to mean that the gelis chemically bound to the pores of the support member. The gel can beheld by the physical constraint imposed upon it by enmeshing andintertwining with structural elements of the support member, withoutactually being chemically grafted to the support member, although insome embodiments, the gel may be grafted to the surface of the pores ofthe support member.

In certain embodiments, the cross-linked gels are macroporous. In theseinstances, because the macropores are present in the gel that occupiesthe pores of the support member, the macropores of the gel must besmaller than the pores of the support member. Consequently, the flowcharacteristics and separation characteristics of the composite materialare dependent on the characteristics of the gel, but are largelyindependent of the characteristics of the porous support member, withthe proviso that the size of the pores present in the support member isgreater than the size of the macropores of the gel. The porosity of thecomposite material can be tailored by filling the support member with agel whose porosity is partially or completely dictated by the nature andamounts of monomer or polymer, cross-linking agent, reaction solvent,and porogen, if used. Properties of the composite material aredetermined partially, if not entirely, by the properties of the gel. Thenet result is that the invention provides control over macropore-size,permeability and surface area of the composite materials.

When present, the number of macropores in the composite material is notdictated by the number of pores in the support material. The number ofmacropores in the composite material can be much greater than the numberof pores in the support member because the macropores are smaller thanthe pores in the support member. As mentioned above, the effect of thepore-size of the support material on the pore-size of the macroporousgel is generally negligible. An exception is found in those cases wherethe support member has a large difference in pore-size and pore-sizedistribution, and where a macroporous gel having very small pore-sizesand a narrow range in pore-size distribution is sought. In these cases,large variations in the pore-size distribution of the support member areweakly reflected in the pore-size distribution of the macroporous gel.In certain embodiments, a support member with a somewhat narrowpore-size range may be used in these situations.

In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the composite materials arerelatively non-toxic.

Preparation of Composite Materials

In certain embodiments, the composite materials of the invention may beprepared by single-step methods. In certain embodiments, these methodsmay use water or other environmentally benign solvents as the reactionsolvent. In certain embodiments, the methods may be rapid and,therefore, may lead to simple and/or rapid manufacturing processes. Incertain embodiments, preparation of the composite materials may beinexpensive.

In certain embodiments, the composite materials may be prepared bymixing a monomer or monomers, a cross-linking agent or agents, aninitiator or initiators, and optionally one or more porogens, in one ormore suitable solvents. In certain embodiments, the resulting mixturemay be homogeneous. In certain embodiments, the mixture may beheterogeneous. In certain embodiments, the mixture may then beintroduced into a suitable porous support, where a gel forming reactionmay take place.

In certain embodiments, a porogen may be added to the reactant mixture,wherein porogens may be broadly described as pore-generating additives.In certain embodiments, the porogen may be selected from the groupconsisting of thermodynamically poor solvents and extractable polymers(e.g., poly(ethyleneglycol)), surfactants, and salts.

In some embodiments, the gel forming reaction must be initiated. Incertain embodiments, the gel forming reaction may be initiated by anyknown method, for example, through thermal activation or exposure to UVradiation. In certain embodiments, the reaction may be initiated by UVradiation in the presence of a photoinitiator. In certain embodiments,the photoinitiator may be selected from the group consisting of2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl-1-propanone (Irgacure2959), 4,4′-azobis(4-cyanovaleric acid) (ACVA),2,2-dimethoxy-2-phenylacetophenone (DMPA), benzophenone, benzoin andbenzoin ethers, such as benzoin ethyl ether and benzoin methyl ether,dialkoxyacetophenones, hydroxyalkylphenones, and α-hydroxymethyl benzoinsulfonic esters. Thermal activation may require the addition of athermal initiator. In certain embodiments, the thermal initiator may beselected from the group consisting of1,1′-azobis(cyclohexanecarbonitrile) (VAZO® catalyst 88),azobis(isobutyronitrile) (AIBN), potassium persulfate, ammoniumpersulfate, and benzoyl peroxide.

In certain embodiments, the gel-forming reaction may be initiated by UVradiation. In certain embodiments, a photoinitiator may be added to thereactants of the gel forming reaction, and the support member containingthe mixture of monomer, cross-linking agent, and photoinitiator may beexposed to UV radiation at wavelengths from about 250 nm to about 400 nmfor a period of a few seconds to a few hours. In certain embodiments,the support member containing the mixture of monomer, cross-linkingagent, and photoinitiator may be exposed to UV radiation at about 350 nmfor a period of a few seconds to a few hours. In certain embodiments,the support member containing the mixture of monomer, cross-linkingagent, and photoinitiator may be exposed to UV radiation at about 350 nmfor about 10 minutes. In certain embodiments, visible wavelength lightmay be used to initiate the polymerization. In certain embodiments, thesupport member must have a low absorbance at the wavelength used so thatthe energy may be transmitted through the support member.

In certain embodiments, the rate at which polymerization is carried outmay have an effect on the size of the macropores obtained in themacroporous gel. In certain embodiments, when the concentration ofcross-linker in a gel is increased to sufficient concentration, theconstituents of the gel begin to aggregate to produce regions of highpolymer density and regions with little or no polymer, which latterregions are referred to as “macropores” in the present specification.This mechanism is affected by the rate of polymerization.

In certain embodiments, once the composite materials are prepared, theymay be washed with various solvents to remove any unreacted componentsand any polymer or oligomers that are not anchored within the support.In certain embodiments, solvents suitable for the washing of thecomposite material include water, acidic (e.g., HCl) or basic (e.g.,NaOH) aqueous solution, aqueous salt solutions (e.g., NaCl), acetone,methanol, ethanol, propanol, and DMF.

Exemplary Uses of the Composite Materials

In certain embodiments, the invention relates to a method, wherein afluid is passed through the cross-linked gel of any one of theaforementioned composite materials. By tailoring the conditions forbinding or fractionation, good selectivity can be obtained.

In certain embodiments, the invention relates to a method of separatingbiomolecules, such as proteins or immunoglobulins, from solution. Incertain embodiments, the invention relates to a method of purifyingbiomolecules, such as proteins or immunoglobulins. In certainembodiments, the invention relates to a method of purifying proteins ormonoclonal antibodies with high selectivity. In certain embodiments, theinvention relates to a method, wherein the biological molecule orbiological ion retains its tertiary or quaternary structure, which maybe important in retaining biological activity. In certain embodiments,biological molecules or biological ions that may be separated orpurified include proteins, such as albumins, e.g., bovine serum albumin,and lysozyme. In certain embodiments, biological molecules or biologicalions that may be separated include γ-globulins of human and animalorigins, immunoglobulins such as IgG, IgM, or IgE of human and animalorigins, proteins of recombinant and natural origin including protein A,phytochrome, halophilic protease, poly(3-hydroxybutyrate) depolymerase,aculaecin-A acylase, polypeptides of synthetic and natural origin,interleukin-2 and its receptor, enzymes such as phosphatase,dehydrogenase, ribonuclease A, etc., monoclonal antibodies, fragments ofantibodies, trypsin and its inhibitor, albumins of varying origins,e.g., α-lactalbumin, human serum albumin, chicken egg albumin, ovalbuminetc., cytochrome C, immunoglobulins, myoglobulin, recombinant humaninterleukin, recombinant fusion protein, nucleic acid derived products,DNA and RNA of synthetic and natural origin, DNA plasmids, lectin,α-chymotrypsinogen, and natural products including small molecules. Incertain embodiments, the invention relates to a method of recovering anantibody fragment from variants, impurities, or contaminants associatedtherewith. In certain embodiments, biomolecule separation orpurification may occur substantially in the cross-linked gel. In certainembodiments, biomolecule separation or purification may occursubstantially in the macropores of the cross-linked gel, when thecross-linked gel has macropores.

In certain embodiments, the invention relates to a method of reversibleadsorption of a substance. In certain embodiments, an adsorbed substancemay be released by changing the liquid that flows through the gel. Incertain embodiments, the uptake and release of substances may becontrolled by variations in the composition of the cross-linked gel.

In certain embodiments, the invention relates to a method, wherein thesubstance may be applied to the composite material from a bufferedsolution.

In certain embodiments, the invention relates to a method, wherein thesubstance may be eluted using varying concentrations and pHs of aqueoussalt solutions.

In certain embodiments, the invention relates to a method that exhibitshigh binding capacities. In certain embodiments, the invention relatesto a method that exhibits binding capacities of about 1mg/mL_(membrane), about 2 mg/mL_(membrane), about 3 mg/mL_(membrane),about 4 mg/mL_(membrane), about 5 mg/mL_(membrane), about 6mg/mL_(membrane), about 7 mg/mL_(membrane), about 8 mg/mL_(membrane),about 9 mg/mL_(membrane), about 10 mg/mL_(membrane), about 12mg/mL_(membrane), about 14 mg/mL_(membrane), about 16 mg/mL_(membrane),about 18 mg/mL_(membrane), about 20 mg/mL_(membrane), about 30mg/mL_(membrane), about 40 mg/mL_(membrane), about 50 mg/mL_(membrane),about 60 mg/mL_(membrane), about 70 mg/mL_(membrane), about 80mg/mL_(membrane), about 90 mg/mL_(membrane), about 100 mg/mL_(membrane),about 110 mg/mL_(membrane), about 120 mg/mL_(membrane), about 130mg/mL_(membrane), about 140 mg/mL_(membrane), about 150mg/mL_(membrane), about 160 mg/mL_(membrane), about 170mg/mL_(membrane), about 180 mg/mL_(membrane), about 190mg/mL_(membrane), about 200 mg/mL_(membrane), about 210mg/mL_(membrane), about 220 mg/mL_(membrane), about 230mg/mL_(membrane), about 240 mg/mL_(membrane), about 250mg/mL_(membrane), about 260 mg/mL_(membrane), about 270mg/mL_(membrane), about 280 mg/mL_(membrane), about 290mg/mL_(membrane), about 300 mg/mL_(membrane), about 320mg/mL_(membrane), about 340 mg/mL_(membrane) mg/mL_(membrane), about 360mg/mL_(membrane), about 380 mg/mL_(membrane), or about 400mg/mL_(membrane) at 10% breakthrough.

The water flux, Q_(H2O) (kg/m² h), was calculated using the followingequation:

$Q_{H_{2}O} = \frac{\left( {m_{1} - m_{2}} \right)}{A \cdot t}$where m₁ is the mass of water transferred through the membrane at t₁, m₂is the mass of water transferred through the membrane at t₂, A is themembrane cross-sectional area and t is the time (where t₁>t₂).

In certain embodiments, an additive may be added to the eluting saltsolution (the second fluid, or the third or later fluid). In certainembodiments, the additive is added in a low concentration (e.g., lessthan about 2 M, about 1 M, about 0.5 M, or about 0.2 M). In certainembodiments, the additive is a water-miscible alcohol, a detergent,dimethyl sulfoxide, dimethyl formamide, or an aqueous solution of achaotropic salt.

In certain embodiments, changing pH is an effective elution tool forprotein elution with or without changing the conductivity of the mobilephase.

Pore Size Determination

SEM and ESEM

As mentioned above, in certain embodiments, the cross-linked gel is amacroporous cross-linked gel. The average diameter of the macropores inthe macroporous cross-linked gel may be estimated by one of manymethods. One method that may be employed is scanning electron microscopy(SEM). SEM is a well-established method for determining pore sizes andporosities in general, and for characterizing membranes in particular.Reference is made to the book Basic Principles of Membrane Technology byMarcel Mulder (© 1996) (“Mulder”), especially Chapter IV. Mulderprovides an overview of methods for characterizing membranes. For porousmembranes, the first method mentioned is electron microscopy. SEM is avery simple and useful technique for characterising microfiltrationmembranes. A clear and concise picture of the membrane can be obtainedin terms of the top layer, cross-section and bottom layer. In addition,the porosity and pore size distribution can be estimated from thephotographs.

Environmental SEM (ESEM) is a technique that allows for thenon-destructive imaging of specimens that are wet, by allowing for agaseous environment in the specimen chamber. The environmental secondarydetector (ESD) requires a gas background to function and operates atfrom about 3 torr to about 20 torr. These pressure restraints limit theability to vary humidity in the sample chamber. For example, at 10 torr,the relative humidity at a specific temperature is as follows:

Relative Humidity at 10 torr (%) T (° C.) About 80 About 16 About 70About 18 About 60 About 20 About 40 About 24 About 20 About 40 About 10About 50 About 2 About 70 About 1 About 100This is a useful guide to relative humidity in the sample chamber atdifferent temperatures. In certain embodiments, the relative humidity inthe sample chamber during imaging is from about 1% to about 99%. Incertain embodiments, the relative humidity in the sample chamber duringimaging is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%,about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%,about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about90%, about 95%, or about 99%. In certain embodiments, the relativehumidity in the sample chamber during imaging is about 45%

In certain embodiments, the microscope has nanometer resolution and upto about 100,000× magnification.

In certain embodiments, the temperature in the sample chamber duringimaging is from about 1° C. to about 95° C. In certain embodiments, thetemperature in the sample chamber during imaging is about 2° C., about3° C., about 4° C., about 5° C., about 6° C., about 7° C., about 8° C.,about 9° C., about 10° C., about 12° C., about 14° C., about 16° C.,about 18° C., about 20° C., about 25° C., about 30° C., about 35° C.,about 40° C., about 45° C., about 50° C., about 55° C., about 60° C.,about 65° C., about 70° C., about 75° C., about 80° C., or about 85° C.In certain embodiments, the temperature in the sample chamber duringimaging is about 5° C.

In certain embodiments, the pressure in the sample chamber duringimaging is from about 0.5 torr to about 20 torr. In certain embodiments,the pressure in the sample chamber during imaging is about 4 torr, about6 torr, about 8 torr, about 10 torr, about 12 torr, about 14 torr, about16 torr, about 18 torr, or about 20 torr. In certain embodiments, thepressure in the sample chamber during imaging is about 3 torr.

In certain embodiments, the working distance from the source of theelectron beam to the sample is from about 6 mm to about 15 mm. Incertain embodiments, the working distance from the source of theelectron beam to the sample is about 6 mm, about 7 mm, about 8 mm, about9 mm, about 10 mm, about 11 mm, about 12 mm, about 13 mm, about 14 mm,or about 15 mm. In certain embodiments, the working distance from thesource of the electron beam to the sample is about 10 mm.

In certain embodiments, the voltage is from about 1 kV to about 30 kV.In certain embodiments, the voltage is about 2 kV, about 4 kV, about 6kV, about 8 kV, about 10 kV, about 12 kV, about 14 kV, about 16 kV,about 18 kV, about 20 kV, about 22 kV, about 24 kV, about 26 kV, about28 kV, or about 30 kV. In certain embodiments, the voltage is about 20kV.

In certain embodiments, the average pore diameter may be measured byestimating the pore diameters in a representative sample of images fromthe top or bottom of a composite material. One of ordinary skill in theart will recognize and acknowledge various experimental variablesassociated with obtaining an ESEM image of a wetted membrane, and willbe able to design an experiment accordingly.

Capillary Flow Porometry

Capillary flow porometry is an analytical technique used to measure thepore size(s) of porous materials. In this analytical technique, awetting liquid is used to fill the pores of a test sample and thepressure of a non-reacting gas is used to displace the liquid from thepores. The gas pressure and flow rate through the sample is accuratelymeasured and the pore diameters are determined using the followingequation: The gas pressure required to remove liquid from the pores isrelated to the size of the pore by the following equation:D=4×γ·cos θ/PD=pore diameterγ=liquid surface tensionθ=liquid contact angleP=differential gas pressureThis equation shows that the pressure required to displace liquid fromthe wetted sample is inversely related to the pore size. Since thistechnique involves the flow of a liquid from the pores of the testsample under pressure, it is useful for the characterization of “throughpores” (interconnected pores that allow fluid flow from one side of thesample to the other). Other pore types (closed and blind pores) are notdetectable by this method.

Capillary flow porometry detects the presence of a pore when gas startsflowing through that pore. This occurs only when the gas pressure ishigh enough to displace the liquid from the most constricted part of thepore. Therefore, the pore diameter calculated using this method is thediameter of the pore at the most constricted part and each pore isdetected as a single pore of this constricted diameter. The largest porediameter (called the bubble point) is determined by the lowest gaspressure needed to initiate flow through a wet sample and a mean porediameter is calculated from the mean flow pressure. In addition, boththe constricted pore diameter range and pore size distribution may bedetermined using this technique.

This method may be performed on small membrane samples (e.g., about 2.5cm diameter) that are immersed in a test fluid (e.g., water, buffer,alcohol). The range of gas pressure applied can be selected from about 0to about 500 psi.

Other Methods of Determining Pore Diameter

Mulder describes other methods of characterizing the average pore sizeof a porous membrane, including atomic force microscopy (AFM) (page164), permeability calculations (page 169), gas adsorption-desorption(page 173), thermoporometry (page 176), permporometry (page 179), andliquid displacement (page 181). Mulder, and the references citedtherein, are hereby incorporated by reference.

Exemplary Composite Materials

In certain embodiments, the invention relates to a composite material,comprising:

a support member, comprising a plurality of pores extending through thesupport member; and

a cross-linked gel, wherein the cross-linked gel comprises a polymerderived from a first monomer and a first cross-linker;

wherein

the cross-linked gel is located in the pores of the support member;

the first monomer comprises two thiol functional groups; and

the first cross-linker comprises (i) at least three carbon-carbon doublebonds, (ii) at least two carbon-carbon triple bonds, or (iii) at leastone carbon-carbon triple bond and at least one carbon-carbon doublebond.

In certain embodiments, the invention relates to a composite material,comprising:

a support member, comprising a plurality of pores extending through thesupport member; and

a cross-linked gel, wherein the cross-linked gel comprises a polymerderived from a first monomer, a second monomer, and a firstcross-linker;

wherein

the cross-linked gel is located in the pores of the support member;

the first monomer comprises two thiol functional groups;

the second monomer comprises two carbon-carbon double bonds; and

the first cross-linker comprises (i) at least three thiol functionalgroups, (ii) at least three carbon-carbon double bonds, (iii) at leasttwo carbon-carbon triple bonds, or (iv) at least one carbon-carbontriple bond and at least one carbon-carbon double bond.

In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the cross-linked gel ismacroporous.

In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the first monomer comprisestwo terminal thiol functional groups.

In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the first monomer issubstantially soluble in DMAc or DPMA, or a mixture thereof.

In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the first monomer is2,2′-(ethylenedioxy)diethanethiol (EDDET), 1,2-ethanedithiol,1,4-butanedithiol, PEG dithiol (such as linear PEG dithiol),1,6-hexanedithiol, 2,2′-thiodiethanethiol, ethane-1,2-diylbis(3-mercaptopropanoate), hexa(ethylene glycol) dithiol, tetra(ethyleneglycol) dithiol, 1,16-hexadecanedithiol, benzene-1,2-dithiol,benzene-1,3-dithiol, benzene-1,4-dithiol, biphenyl-4,4′-dithiol,p-terphenyl-4,4″-dithiol, (S)-2-aminobutane-1,4-dithiol hydrochloride,4-phenyl-4H-(1,2,4)tri azole-3,5-dithiol,5-(4-chlorophenyl)-pyrimidine-4,6-dithiol, 1,4-benzenedimethanethiol,2-mercaptoethyl ether, or L-(−)-dithiothreitol.

In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the second monomer comprisestwo terminal carbon-carbon double bonds.

In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the second monomer issubstantially soluble in DMAc or DPMA, or a mixture thereof.

In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the second monomer istri(ethylene glycol) divinyl ether (TEGDV), 1,9-decadiene,1,4-bis(vinyloxy)butane, diallylphthalate, diallyl diglycol carbonate,poly(ethylene glycol) divinyl ether, divinyl glycol, or divinylbenzene,divinyl sulfone, 1,4-butanediol divinyl ether, allyl ether, allylsulfide, 1,4-bis(4-vinylphenoxy)butane, 1,5-hexadiene, dipentene,(R)-(+)-limonene, (S)-(−)-limonene, N,N′-methylenebis(acrylamide), orN,N′-ethylenebis(acrylamide).

In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the mole ratio of firstmonomer to second monomer is greater than 1:1, for example about 1.5:1,about 2:1, about 2.5:1, about 3:1, about 3.5:1, about 4:1, about 4.5:1,or about 5:1.

In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the composite materialfurther comprises a third monomer, wherein the third monomer comprisestwo carbon-carbon double bonds.

In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the third monomer comprisestwo terminal carbon-carbon double bonds.

In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the third monomer issubstantially soluble in DMAc or DPMA, or a mixture thereof.

In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the third monomer is(+)-N,N′-diallyltartramide (DATA), diallyl disulfide, diallyl carbonate,diallyl maleate, diallyl succinate, trimethylolpropane diallyl ether,1,1-diallyl-1-docosanol, 1,1-diallyl-3-(1-naphthyl)urea,1,1-diallyl-3-(2-ethylphenyl)urea, 1,2-diallyl-1,2-cyclohexanediol,2,6-diallyl-meta-cresol, N,N-diallyl-2-hydroxypropanamide,1,4-pentadien-3-ol, trimethyl(propargyl)silane, or propargylamine.

In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the mole ratio of firstmonomer to third monomer is from greater than 1:1, for example about1.5:1, about 2:1, about 2.5:1, about 3:1, about 3.5:1, about 4:1, about4.5:1, or about 5:1.

In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the first cross-linkercomprises at least three carbon-carbon double bonds.

In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the first cross-linkercomprises three carbon-carbon double bonds. In certain embodiments, theinvention relates to any one of the aforementioned composite materials,wherein the first cross-linker comprises three terminal carbon-carbondouble bonds.

In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the first cross-linkercomprises two carbon-carbon triple bonds. In certain embodiments, theinvention relates to any one of the aforementioned composite materials,wherein the first cross-linker comprises two terminal carbon-carbontriple bonds.

In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the first cross-linker issubstantially soluble in DMAc or DPMA, or a mixture thereof.

In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the first cross-linker is1,3,5-triallyl-1,3, 5-triazine-2,4,6(1H,3H,5H)-trione (TATATO),1,6-heptadiyne, 1,7-octadiyne, 1,8-nonadiyne, 1,9-decadiyne, propargylacrylate, 4-arm PEG norbornene (Fairbanks, B. D., et al. Adv. Mater.2009, 21 (48), 5005-5010), trimethylolpropane triacrylate, tetra-alkynepoly(ethylene glycol) (e.g., Daniele, M. A., et al. Biomaterials 2014,35, 1845-1856), 2,4,6-triallyloxy-1,3,5-triazine, triallylamine,triallyl borate, triallylphosphine, diallyl fumarate,3-(allyloxy)-1-propyne, glyoxal bis(diallyl acetal), tetraallylsilane,propargyl ether, or squalene.

In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the first cross-linker istrimethylolpropanetri(3-mercaptopropionate), pentaerythritoltetra(3-mercaptopropionate), poly(ethylene glycol) tetra-thiol (e.g.,Daniele, M. A., et al. Biomaterials 2014, 35, 1845-1856),tris[2(3-mercaptopropionyl oxy)ethyl]isocyanurate, pentaerythritoltetrakis(2-mercaptoacetate), trithiocyanuric acid, or 1-thiohexitol.

In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the composite materialfurther comprises a second cross-linker; and the second cross-linkercomprises (i) at least three thiol functional groups, (ii) at leastthree carbon-carbon double bonds, (iii) at least two carbon-carbontriple bonds, or (iv) at least one carbon-carbon triple bond and atleast one carbon-carbon double bond.

In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the second cross-linkercomprises at least two carbon-carbon triple bonds.

In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the second cross-linkercomprises two carbon-carbon triple bonds. In certain embodiments, theinvention relates to any one of the aforementioned composite materials,wherein the second cross-linker comprises two terminal carbon-carbontriple bonds.

In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the second cross-linker isdifferent from the first cross-linker.

In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the second cross-linker issubstantially soluble in DMAc or DPMA, or a mixture thereof.

In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the second cross-linker is1,3,5-triallyl-1,3,5-triazine-2,4,6(1H,3H,5H)-trione (TATATO),1,6-heptadiyne, 1,7-octadiyne, 1,8-nonadiyne, 1,9-decadiyne, propargylacrylate, 2,4,6-triallyloxy-1,3,5-triazine, triallylamine, triallylborate, triallylphosphine, diallyl fumarate, 3-(allyloxy)-1-propyne,dipropargylamine, 5,6-dimethyl-5-decen-1,9-diyne, glyoxal bis(diallylacetal), tetraallyl silane, propargyl ether, squalene,trimethylolpropanetri(3-mercaptopropionate), pentaerythritoltetra(3-mercaptopropionate), poly(ethylene glycol) tetra-thiol,tris[2(3-mercaptopropionyloxy)ethyl]isocyanurate, pentaerythritoltetrakis(2-mercaptoacetate) trithiocyanuric acid, or 1-thiohexitol.

In certain embodiments, the invention relates to a composite material,comprising:

a support member, comprising a plurality of pores extending through thesupport member; and

a cross-linked gel, wherein the cross-linked gel comprises a polymerderived from a first monomer and a first cross-linker;

wherein

the cross-linked gel is located in the pores of the support member;

the first monomer comprises (i) two carbon-carbon double bonds, or (ii)a carbon-carbon triple bond; and

the first cross-linker comprises at least three thiol functional groups.

In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the cross-linked gel ismacroporous.

In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the first monomer comprisestwo terminal carbon-carbon double bonds.

In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the first monomer issubstantially soluble in DMAc or DPMA, or a mixture thereof.

In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the first monomer istri(ethylene glycol) divinyl ether (TEGDV), 1,9-decadiene,1,4-bis(vinyloxy)butane, diallylphthalate, diallyl diglycol carbonate,poly(ethylene glycol) divinyl ether, divinyl glycol, or divinylbenzene,divinyl sulfone, 1,4-butanediol divinyl ether, allyl ether, allylsulfide, 1,4-bis(4-vinylphenoxy)butane, 1,5-hexadiene, dipentene,(R)-(+)-limonene, or (S)-(−)-limonene.

In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the mole ratio of firstmonomer to second monomer is greater than 1:1, for example about 1.5:1,about 2:1, about 2.5:1, about 3:1, about 3.5:1, about 4:1, about 4.5:1,or about 5:1.

In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the first cross-linker issubstantially soluble in DMAc or DPMA, or a mixture thereof.

In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the first cross-linker istrimethylolpropanetri(3-mercaptopropionate), pentaerythritoltetra(3-mercaptopropionate), poly(ethylene glycol) tetra-thiol (e.g.,Daniele, M. A., et al. Biomaterials 2014, 35, 1845-1856),tris[2(3-mercaptopropionyloxy)ethyl]isocyanurate, pentaerythritoltetrakis(2-mercaptoacetate), trithiocyanuric acid, or 1-thiohexitol.

In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the cross-linked gel furthercomprises a plurality of grafted end-groups. In certain embodiments, theinvention relates to any one of the aforementioned composite materials,wherein the grafted end-groups are derived from a molecule having athiol functional group or a molecule having an unsaturated carbon-carbonbond.

In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the grafted end-groups arederived from a molecule having a thiol functional group or a moleculehaving an unsaturated carbon-carbon bond; and the molecule having athiol functional group or the molecule having an unsaturatedcarbon-carbon bond has a log P from about 0.5 to about 8.0. In certainembodiments, composite materials having hydrophobic grafted end-groupsare useful for hydrophobic interaction chromatography.

In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the molecule having a thiolfunctional group and the molecule having an unsaturated carbon-carbonbond are substantially soluble in DMAc or DPMA, or a mixture thereof.

In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the grafted end-groups arederived from a molecule having a thiol functional group; and themolecule having a thiol functional group is 3-mercaptopropionic acid,1-mercaptosuccinic acid, a peptide having a cysteine residue, a proteinhaving a cysteine residue (either a naturally occurring cysteine residueor an engineered cysteine residue, e.g., Protein A), cysteamine,1-thiohexitol, poly(ethylene glycol) 2-mercaptoethyl ether acetic acid,poly(ethylene glycol) methyl ether thiol, 1-thioglycerol,2-naphthalenethiol, biphenyl-4-thiol, 3-amino-1,2,4-triazole-5-thiol,5-(trifluoromethyl)pyridine-2-thiol,1-[2-(dimethylamino)ethyl]-1H-tetrazole-5-thiol, 1-propanethiol,1-butanethiol, 1-pentanethiol, 1-hexanethiol, 1-octanethiol,8-amino-1-octanethiol hydrochloride,3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluoro-1-octanethiol,8-mercapto-1-octanol, or γ-Glu-Cys.

In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the grafted end-groups arederived from a molecule having an unsaturated carbon-carbon bond; andthe molecule having an unsaturated carbon-carbon bond is 1-octene,1-hexyne, 4-bromo-1-butene, allyldiphenylphosphine, allylamine, allylalcohol, 3,4-dihydroxy-1-butene, 7-octene-1,2-diol,3-allyloxy-1,2-propanediol, 3-butenoic acid, 3,4-dehydro-L-proline,vinyl laurate, 1-vinyl-2-pyrrolidinone, vinyl cinnamate, an acylamide,or an acrylate.

In certain embodiments, the invention relates to any one of theaforementioned composite materials wherein the cross-linked gelcomprises macropores; and the macropores have an average pore diameterof about 10 nm to about 3000 nm. In certain embodiments, the diameter ofthe macropores is estimated by one of the techniques described herein.In certain embodiments, the diameter of the macropores is calculated bycapillary flow porometry.

In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the average pore diameter ofthe macropores is about 25 nm to about 1500 nm.

In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the average pore diameter ofthe macropores is about 50 nm to about 1000 nm. In certain embodiments,the invention relates to any one of the aforementioned compositematerials, wherein the average pore diameter of the macropores is about50 nm, about 100 nm, about 150 nm, about 200 nm, about 250 nm, about 300nm, about 350 nm, about 400 nm, about 450 nm, about 500 nm, about 550nm, about 600 nm, about 650 nm, or about 700 nm.

In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the average pore diameter ofthe macropores is about 300 nm to about 400 nm.

In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the composite material is amembrane.

In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the support member has avoid volume; and the void volume of the support member is substantiallyfilled with the macroporous cross-linked gel.

In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the support member comprisesa polymer; the support member is about 10 μm to about 1000 μm thick; thepores of the support member have an average pore diameter of about 0.1μm to about 25 μm. In certain embodiments, the support member has avolume porosity of about 40% to about 90%.

In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the thickness of the supportmember is about 10 μm to about 1000 μm. In certain embodiments, theinvention relates to any one of the aforementioned composite materials,wherein the thickness of the support member is about 10 μm to about 500μm. In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the thickness of the supportmember is about 30 μm to about 300 μm. In certain embodiments, theinvention relates to any one of the aforementioned composite materials,wherein the thickness of the support member is about 30 μm, about 50 μm,about 100 μm, about 150 μm, about 200 μm, about 250 μm, or about 300 μm.

In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the pores of the supportmember have an average pore diameter of about 0.1 μm to about 25 μm. Incertain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the pores of the supportmember have an average pore diameter of about 0.5 μm to about 15 μm. Incertain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the pores of the supportmember have an average pore diameter of about 0.5 μm, about 1 μm, about2 μm, about 3 μm, about 4 μm, about 5 μm, about 6 μm, about 7 μm, about8 μm, about 9 μm, about 10 μm, about 11 μm, about 12 μm, about 13 μm,about 14 μm, or about 15 μm.

In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the support member has avolume porosity of about 40% to about 90%. In certain embodiments, theinvention relates to any one of the aforementioned composite materials,wherein the support member has a volume porosity of about 50% to about80%. In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the support member has avolume porosity of about 50%, about 60%, about 70%, or about 80%.

In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the support member comprisesa polyolefin.

In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the support member comprisesa polymeric material selected from the group consisting of polysulfones,polyethersulfones, polyphenyleneoxides, polycarbonates, polyesters,cellulose and cellulose derivatives.

In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the support member comprisesa fibrous woven or non-woven fabric comprising a polymer; the supportmember is from about 10 μm to about 2000 μm thick; the pores of thesupport member have an average pore diameter of from about 0.1 μm toabout 25 μm; and the support member has a volume porosity of about 40%to about 90%.

In certain embodiments, the invention relates to any one of theaforementioned composite materials, wherein the composite material has awater contact angle from about 50° to about 120°.

Exemplary Methods of Use

In certain embodiments, the invention relates to a method, comprisingthe step of:

contacting at a first flow rate a first fluid comprising a substancewith any one of the aforementioned composite materials, therebyadsorbing or absorbing a portion of the substance onto the compositematerial.

In certain embodiments, the first fluid further comprises a fragmentedantibody, aggregated antibodies, a host cell protein, a polynucleotide,an endotoxin, or a virus.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the fluid flow path of the first fluidis substantially through the macropores of the composite material.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the fluid flow path of the first fluidis substantially perpendicular to the macropores of the compositematerial.

In certain embodiments, the invention relates to any one of theaforementioned methods, further comprising the step of:

contacting at a second flow rate a second fluid with the substanceadsorbed or absorbed onto the composite material, thereby releasing afirst portion of the substance from the composite material.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the fluid flow path of the second fluidis substantially through the macropores of the composite material.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the fluid flow path of the second fluidis substantially perpendicular to the macropores of the compositematerial.

In certain embodiments, the invention relates to any one of theaforementioned methods, further comprising the step of:

contacting at a third flow rate a third fluid with the substanceadsorbed or absorbed onto the composite material, thereby releasing asecond portion of the substance from the composite material.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the substance is a biological molecule,biological ion, virus, or virus particle.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the substance is a biological moleculeor biological ion selected from the group consisting of albumins,lysozyme, viruses, cells, γ-globulins of human and animal origins,immunoglobulins of human and animal origins, proteins of recombinant andnatural origins, polypeptides of synthetic and natural origins,interleukin-2 and its receptor, enzymes, monoclonal antibodies, trypsinand its inhibitor, cytochrome C, myoglobin, myoglobulin,α-chymotrypsinogen, recombinant human interleukin, recombinant fusionprotein, nucleic acid derived products, DNA of synthetic and naturalorigins, and RNA of synthetic and natural origins.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the biological molecule or biologicalion is lysozyme, hIgG, myoglobin, human serum albumin, soy trypsininhibitor, transferring, enolase, ovalbumin, ribonuclease, egg trypsininhibitor, cytochrome c, Annexin V, or α-chymotrypsinogen.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the first fluid is a buffer. In certainembodiments, the invention relates to any one of the aforementionedmethods, wherein the concentration of the buffer in the first fluid isabout 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30mM, about 35 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM,about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about0.1 M, about 0.11 M, about 0.12 M, about 0.13 M, about 0.14 M, about0.15 M, about 0.16 M, about 0.17 M, about 0.18 M, about 0.19 M or about0.2 M. In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the pH of the first fluid is about 5,about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5,or about 9.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the first fluid comprises sodiumphosphate.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the first fluid comprises a salt. Incertain embodiments, the invention relates to any one of theaforementioned methods, wherein the concentration of the salt in thefirst fluid is about about 50 mM, about 60 mM, about 70 mM, about 75 mM,about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 0.1 M, about0.11 M, about 0.12 M, about 0.13 M, about 0.14 M, about 0.15 M, about0.16 M, about 0.17 M, about 0.18 M, about 0.19 M about 0.2 M, about 0.25M, or about 0.3 M. In certain embodiments, the invention relates to anyone of the aforementioned methods, wherein the salt is sodium chloride.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the concentration of the substance inthe first fluid is about 0.2 mg/mL to about 10 mg/mL. In certainembodiments, the invention relates to any one of the aforementionedmethods, wherein the concentration of the substance in the first fluidis about 0.2 mg/mL, about 0.3 mg/mL, about 0.4 mg/mL, about 0.5 mg/mL,about 0.6 mg/mL, about 0.7 mg/mL, about 0.8 mg/mL, about 0.9 mg/L, about1 mg/mL, about 1.2 mg/mL, about 1.4 mg/mL, about 1.6 mg/mL, about 1.8mg/mL, about 2 mg/mL, about 3 mg/mL, about 4 mg/mL, about 5 mg/mL, about6 mg/mL, about 7 mg/mL, about 8 mg/mL, about mg/mL, or about 10 mg/mL.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the first flow rate is about 3 membranevolumes (MV)/min to about 70 MV/min. In certain embodiments, theinvention relates to any one of the aforementioned methods, wherein thefirst flow rate is about 5 MV/min to about 30 MV/min. In certainembodiments, the invention relates to any one of the aforementionedmethods, wherein the first flow rate is about 10 MV/min to about 20MV/min. In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the first flow rate is about 10 MV/min,about 11 MV/min, about 12 MV/min, about 13 MV/min, about 14 MV/min,about 15 MV/min, about 16 MV/min, about 17 MV/min, about 18 MV/min,about 19 MV/min, or about 20 MV/min.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the first flow rate is about 0.5 mL/minto about 50 L/min. In certain embodiments, the invention relates to anyone of the aforementioned methods, wherein the first flow rate is about0.5 mL/min to about 25 L/min. In certain embodiments, the inventionrelates to any one of the aforementioned methods, wherein the first flowrate is about 0.5 mL/min to about 10 L/min. In certain embodiments, theinvention relates to any one of the aforementioned methods, wherein thefirst flow rate is about 0.5 mL/min to about 1 L/min. In certainembodiments, the invention relates to any one of the aforementionedmethods, wherein the first flow rate is about 0.5 mL/min to about 0.5L/min. In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the first flow rate is about 0.5 mL/minto about 100 mL/min. In certain embodiments, the invention relates toany one of the aforementioned methods, wherein the first flow rate isabout 0.5 mL/min to about 10 mL/min. In certain embodiments, theinvention relates to any one of the aforementioned methods, wherein thefirst flow rate is about 0.5 mL/min to about 2 mL/min. In certainembodiments, the invention relates to any one of the aforementionedmethods, wherein the first flow rate is about 0.5 mL/min, about 0.6mL/min, about 0.7 mL/min, about 0.8 mL/min, about 0.9 mL/min, about 1mL/min, about 1.1 mL/min, about 1.2 mL/min, about 1.3 mL/min, about 1.4mL/min, about 1.5 mL/min, about 1.6 mL/min, about 1.7 mL/min, or about1.8 mL/min.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the second fluid is a buffer. In certainembodiments, the invention relates to any one of the aforementionedmethods, wherein the second fluid comprises glycine-HCl or sodiumcitrate. In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the second fluid comprises glycine-HClor sodium citrate in a concentration of about 5 mM to about 2 M. Incertain embodiments, the invention relates to any one of theaforementioned methods, wherein the second fluid comprises glycine-HClor sodium citrate in about 5 mM, about 10 mM, about 20 mM, about 30 mM,about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about90 mM, about 100 mM, about 125 mM, about 150 mM, about 200 mM, about 300mM, or about 400 mM.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the pH of the second fluid is about 2 toabout 8. In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the pH of the second fluid is about 2,about 2.2, about 2.4, about 2.6, about 2.8, about 3, about 3.2, about3.4, about 3.6, about 3.8, about 4, about 4.2, about 4.4, about 4.6,about 4.8, about 5, about 5.2, about 5.4, about 5.5, about 5.6, about5.7, about 5.8, about 5.9, about 6, about 6.1, about 6.2, about 6.3,about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6,about 7.7, about 7.8, about 7.9, or about 8.0.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the second flow rate is about 3 membranevolumes (MV)/min to about 70 MV/min. In certain embodiments, theinvention relates to any one of the aforementioned methods, wherein thesecond flow rate is about 5 MV/min to about 30 MV/min. In certainembodiments, the invention relates to any one of the aforementionedmethods, wherein the second flow rate is about 10 MV/min to about 20MV/min. In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the second flow rate is about 10 MV/min,about 11 MV/min, about 12 MV/min, about 13 MV/min, about 14 MV/min,about 15 MV/min, about 16 MV/min, about 17 MV/min, about 18 MV/min,about 19 MV/min, or about 20 MV/min.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the second flow rate is about 0.5 mL/minto about 50 L/min. In certain embodiments, the invention relates to anyone of the aforementioned methods, wherein the second flow rate is about0.5 mL/min to about 25 L/min. In certain embodiments, the inventionrelates to any one of the aforementioned methods, wherein the secondflow rate is about 0.5 mL/min to about 10 L/min. In certain embodiments,the invention relates to any one of the aforementioned methods, whereinthe second flow rate is about 0.5 mL/min to about 1 L/min. In certainembodiments, the invention relates to any one of the aforementionedmethods, wherein the second flow rate is about 0.5 mL/min to about 0.5L/min. In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the second flow rate is about 0.5 mL/minto about 100 mL/min. In certain embodiments, the invention relates toany one of the aforementioned methods, wherein the second flow rate isabout 0.5 mL/min to about 10 mL/min. In certain embodiments, theinvention relates to any one of the aforementioned methods, wherein thesecond flow rate is about 0.5 mL/min to about 2 mL/min. In certainembodiments, the invention relates to any one of the aforementionedmethods, wherein the second flow rate is about 0.5 mL/min, about 0.6mL/min, about 0.7 mL/min, about 0.8 mL/min, about 0.9 mL/min, about 1mL/min, about 1.1 mL/min, about 1.2 mL/min, about 1.3 mL/min, about 1.4mL/min, about 1.5 mL/min, about 1.6 mL/min, about 1.7 mL/min, or about1.8 mL/min.

In certain embodiments, the invention relates to any one of theaforementioned methods, further comprising the steps of:

cleaning the composite material; and

repeating the above-mentioned steps.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the composite material is cleaned with abasic solution. In certain embodiments, the invention relates to any oneof the aforementioned methods, wherein the composite material is cleanedwith a fourth fluid; and the fourth fluid comprises sodium hydroxide.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein substantially all of the substance isadsorbed or absorbed onto the composite material.

In certain embodiments, the invention relates to a method, comprisingthe step of:

contacting at a first flow rate a first fluid comprising a substance andan unwanted material with any one of the aforementioned compositematerials, thereby adsorbing or absorbing a portion of the unwantedmaterial onto the composite material.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the unwanted material comprises afragmented antibody, aggregated antibodies, a host cell protein, apolynucleotide, an endotoxin, or a virus.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein substantially all of the unwantedmaterial is adsorbed or absorbed onto the composite material.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the fluid flow path of the first fluidis substantially through the macropores of the composite material.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the substance is a biological moleculeor biological ion.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the biological molecule or biologicalion is selected from the group consisting of albumins, lysozyme,viruses, cells, γ-globulins of human and animal origins, immunoglobulinsof human and animal origins, proteins of recombinant and naturalorigins, polypeptides of synthetic and natural origins, interleukin-2and its receptor, enzymes, monoclonal antibodies, trypsin and itsinhibitor, cytochrome C, myoglobin, myoglobulin, α-chymotrypsinogen,recombinant human interleukin, recombinant fusion protein, nucleic acidderived products, DNA of synthetic and natural origins, and RNA ofsynthetic and natural origins.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the biological molecule or biologicalion is lysozyme, hIgG, myoglobin, human serum albumin, soy trypsininhibitor, transferring, enolase, ovalbumin, ribonuclease, egg trypsininhibitor, cytochrome c, Annexin V, or α-chymotrypsinogen.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the first fluid is a clarified cellculture supernatant.

Exemplary Methods of Making

In certain embodiments, the invention relates to a method of making acomposite material, comprising the steps of:

combining a first monomer, a first cross-linker, a photoinitiator, and afirst solvent, wherein the first monomer comprises two thiol functionalgroups; and the first cross-linker comprises (i) at least threecarbon-carbon double bonds, (ii) at least two carbon-carbon triplebonds, or (iii) at least one carbon-carbon triple bond and at least onecarbon-carbon double bond, thereby forming a monomeric mixture;

contacting a support member with the monomeric mixture, thereby forminga modified support member; wherein the support member comprises aplurality of pores extending through the support member, and the averagepore diameter of the pores is about 0.1 to about 25 μm;

covering the modified support member with a polymeric sheet, therebyforming a covered support member; and

irradiating the covered support member for a period of time, therebyforming a composite material.

In certain embodiments, the invention relates to a method of making acomposite material, comprising the steps of:

combining a first monomer, a second monomer, a first cross-linker, aphotoinitiator, and a first solvent, thereby forming a monomericmixture; wherein the first monomer comprises two thiol functionalgroups; the second monomer comprises two carbon-carbon double bonds; andthe first cross-linker comprises (i) at least three thiol functionalgroups, (ii) at least three carbon-carbon double bonds, (iii) at leasttwo carbon-carbon triple bonds, or (iv) at least one carbon-carbontriple bond and at least one carbon-carbon double bond;

contacting a support member with the monomeric mixture, thereby forminga modified support member; wherein the support member comprises aplurality of pores extending through the support member, and the averagepore diameter of the pores is about 0.1 to about 25 μm;

covering the modified support member with a polymeric sheet, therebyforming a covered support member; and

irradiating the covered support member for a period of time, therebyforming a composite material.

In certain embodiments, the invention relates to any one of theaforementioned methods, further comprising the step of washing thecomposite material with a second solvent, thereby forming a washedcomposite material. In certain embodiments, the second solvent is water.

In certain embodiments, the invention relates to any one of theaforementioned methods, further comprising the step of removing anyexcess monomeric mixture from the covered support member.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the composite material is any one of theaforementioned composite materials.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the monomeric mixture further comprisesa plurality of end-group precursors; and the end-group precursors aremolecules having a thiol functional group or molecules having anunsaturated carbon-carbon bond.

In certain embodiments, the invention relates to any one of theaforementioned methods, further comprising the steps of:

contacting the composite material with a mixture comprising aphotoinitiator and a plurality of end-group precursors, thereby forminga grafting mixture; wherein the end-group precursors are moleculeshaving a thiol functional group or molecules having an unsaturatedcarbon-carbon bond; and

irradiating the grafting mixture for a period of time, thereby forming amodified composite material.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the end-group precursor has a log P fromabout 0.5 to about 8.0.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the end-group precursor is substantiallysoluble in DMAc or DPMA, or a mixture thereof.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the end-group precursor is a moleculehaving a thiol functional group; and the molecule having a thiolfunctional group is 3-mercaptopropionic acid, 1-mercaptosuccinic acid, apeptide having a cysteine residue, a protein having a cysteine residue,cysteamine, 1-thiohexitol, poly(ethylene glycol) 2-mercaptoethyl etheracetic acid, poly(ethylene glycol) methyl ether thiol, 1-thioglycerol,2-naphthalenethiol, biphenyl-4-thiol, 3-amino-1,2,4-triazole-5-thiol,5-(trifluoromethyl)pyridine-2-thiol,1-[2-(dimethylamino)ethyl]-1H-tetrazole-5-thiol, 1-propanethiol,1-butanethiol, 1-pentanethiol, 1-hexanethiol, 1-octanethiol,8-amino-1-octanethiol hydrochloride,3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluoro-1-octanethiol,8-mercapto-1-octanol, or γ-Glu-Cys.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the end-group precursor is derived froma molecule having an unsaturated carbon-carbon bond; and the moleculehaving an unsaturated carbon-carbon bond is 1-octene, 1-hexyne,4-bromo-1-butene, allyldiphenylphosphine, allylamine, allyl alcohol,3,4-dihydroxy-1-butene, 7-octene-1,2-diol, 3-allyloxy-1,2-propanediol,3-butenoic acid, 3,4-dehydro-L-proline, vinyl laurate,1-vinyl-2-pyrrolidinone, vinyl cinnamate, an acylamide, or an acrylate.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the ratio of reactive thiol groups toreactive alkene groups (where an alkyne group is equivalent to twoalkene groups) in the monomeric mixture is from about 1:10 to about 2:1,for example, about 1:10, about 1:9, about 1:8, about 1:7, about 1:6,about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, or about 2:1.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the first monomer is present in themonomeric mixture in an amount from about 5% to about 25% by weight ofthe monomeric mixture. In certain embodiments, the invention relates toany one of the aforementioned methods, wherein the first monomer ispresent in the monomeric mixture in an amount from about 5% to about 20%by weight of the monomeric mixture.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the second monomer is present in themonomeric mixture in an amount from about 0.1% to about 20% by weight ofthe monomeric mixture.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the first cross-linker is present in themonomeric mixture in an amount from about 1% to about 20% by weight ofthe monomeric mixture.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the photoinitiator is present in themonomeric mixture in an amount from about 0.1% to about 2% by weight ofthe monomeric mixture.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the photoinitiator is benzoin or abenzoin ether, benzophenone, a dialkoxyacetophenone,2,2-dimethoxy-2-phenylacetophenone,diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide, a hydroxyalkylphenone,1-hydroxy-cyclohexyl-phenyl-ketone,4-(2-hydroxyethoxy)phenyl-(2-hydroxy-2-propyl)ketone,1-[4-(2-hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propane-1-one,2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl-1-propanone, aα-hydroxymethyl benzoin sulfonic ester, 2-hydroxy-2-methylpropiophenone,lithium acylphospinate, or2-methyl-1-[4-(methylthio)phenyl]-2-(4-morpholinyl)-1-propanone,4,4′-azobis(4-cyanovaleric acid) (ACVA), or a mixture thereof.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the first solvent comprisesN,N′-dimethylacetamide (DMAc), (±)-1,3-butanediol (Budiol), di(propyleneglycol)methyl ether acetate (DPMA), water, di(propylene glycol) dimethylether (DPM), di(propylene glycol) propyl ether (DPGPE), di(propyleneglycol) methyl ether (DPGME), tri(propylene glycol) butyl ether (TPGBE),3-methyl-1,3-butanediol, 3,3-dimethyl-1,2-butanediol,3-methoxy-1-butanol, dimethyl sulfoxide (DMSO), ethylene glycol,di(ethylene glycol), tri(ethylene glycol), tetra(ethylene glycol),hexylene glycol, sodium dodecyl sulfate, or N,N-dimethylformamide (DMF),or a mixture thereof.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein N,N′-dimethylacetamide (DMAc) is presentin the monomeric mixture in an amount from about 0% to about 70% byweight of the monomeric mixture. In certain embodiments, the inventionrelates to any one of the aforementioned methods, whereinN,N′-dimethylacetamide (DMAc) is present in the monomeric mixture in anamount from about 0% to about 50% by weight of the monomeric mixture. Incertain embodiments, the invention relates to any one of theaforementioned methods, wherein N,N′-dimethylacetamide (DMAc) is presentin the monomeric mixture in an amount from about 0% to about 70% byweight of the total solvents. In certain embodiments, the inventionrelates to any one of the aforementioned methods, whereinN,N′-dimethylacetamide (DMAc) is present in the monomeric mixture in anamount from about 0% to about 50% by weight of the total solvents.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein (±)-1,3-butanediol (Budiol) is presentin the monomeric mixture in an amount from about 0% to about 50% byweight of the monomeric mixture. In certain embodiments, the inventionrelates to any one of the aforementioned methods, wherein(±)-1,3-butanediol (Budiol) is present in the monomeric mixture in anamount from about 0% to about 50% by weight of the total solvents.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein di(propylene glycol)methyl ether acetate(DPMA) is present in the monomeric mixture in an amount from about 0% toabout 60% by weight of the monomeric mixture. In certain embodiments,the invention relates to any one of the aforementioned methods, whereindi(propylene glycol)methyl ether acetate (DPMA) is present in themonomeric mixture in an amount from about 0% to about 60% by weight ofthe total solvents.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein water is present in the monomericmixture in an amount from about 0% to about 50% by weight of themonomeric mixture. In certain embodiments, the invention relates to anyone of the aforementioned methods, wherein water is present in themonomeric mixture in an amount from about 0% to about 30% by weight ofthe monomeric mixture. In certain embodiments, the invention relates toany one of the aforementioned methods, wherein water is present in themonomeric mixture in an amount from about 0% to about 30% by weight ofthe total solvents.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the covered support member is irradiatedat about 350 nm.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the period of time is about 1 minute,about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes,about 30 minutes, about 45 minutes, or about 1 hour.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the composite material comprisesmacropores.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the average pore diameter of themacropores is less than the average pore diameter of the pores.

EXEMPLIFICATION

The following examples are provided as illustrations. It will beunderstood, however, that the specific details given in each examplehave been selected for purpose of illustration and are not to beconstrued as limiting the scope of the disclosure. Generally, theexperiments were conducted under similar conditions unless noted.

General Materials and Methods

Chemicals:

2,2′-(Ethylenedioxy)diethanethiol (EDDET), 1,4-dithioerythritol (DTT),pentaerythritol tetrakis(3-mercaptopropionate) (PETM),1,3,5-triallyl-1,3,5-triazine-2,4,6(1H,3H,5H)-trione (TATATO),tri(ethylene glycol) divinyl ether (TEGDV), 1,7-octadiyne (OctDi),(+)-N,N′-diallytartramide (DATA), 1-thioglycerol (TG), 1-octanethiol,N,N′-dimethyl acetamide (DMAc), (±)-1,3-butanediol (Budiol),di(propylene glycol)methyl ether acetate (DPMA), ethylene glycol (EG),diethylene glycol (DEG), triethylene glycol (TEG), tetraethylene glycol(TetEG), hexylene glycol, isopropanol, sodium dodecyl sulfate (SDS),4-(2-hydroxyethoxy)phenyl-(2-hydroxy-2-propyl)ketone (IRGACURE 2959),4,4′-azobis(4-cyanovaleric acid) (ACVA), cysteamine hydrochloride,2-mercaptoethanol, mercaptosuccinic acid, sodium phosphate monobasicmonohydrate, potassium phosphate dibasic, potassium phosphate monobasic,sodium acetate trihydrates, glacial acetic acid, sodium hydroxidepellet, glycine, citric acid, and D-(+)-trehalose dehydrate wereobtained from Aldrich.

Proteins:

rProtein A-cys was obtained from Biomedal S.L (Seville, Spain).Polyclonal immuno γ-globulin IgG was obtained from Equitech-Bio Inc.(Kerrville, Tex., USA).

Membrane Preparation:

The crosslinker(s) and monomers (except thiol functionalized monomers,which were added 10 min prior to casting) were added with thephoto-initiator (Irgacure 2959) to a solvent mixture, and the mixturewas stirred long enough to dissolve all components. A pre-weighed 7″×8″porous support substrate sheet (non-woven polypropylene mesh) was placedon a polyethylene sheet, then ˜15 g of the polymer solution was pouredinto the substrate sheet. The impregnated substrate was subsequentlycovered with another polyethylene sheet. The sheet was pressed gently ina circular motion by hand in order to remove excess solution and anyentrapped air bubbles. The polymerization process was initiated byirradiating with UV light (˜350 nm) the polymer solution/substratesandwiched between polyethylene sheets in a closed chamber for 10 min.The resultant membrane was then removed from between the polyethylenesheets and subjected to extensive washing cycles that involved 20-30minutes soaking periods in purified (RO) water (2-3 times) withagitation. The clean membranes were dried by hanging freely in the airat room temperature for ˜16 hours.

Mass Gain, Wetting, and Permeability of Composite Membranes

The weight of the dried membrane was measured and used to calculate themass gain. Wetting of the membrane was also determined by dispensing a50 μL drop of distilled water on the membrane surface and measuring thetime required for the drop to be absorbed within the membrane. Toestimate membrane permeability, the flux of each membrane was determinedusing RO water (or acetate buffer pH 5) and a 7.7-cm diameter membranesample, using 100 kPa applied pressure.

To estimate membrane permeability, the flux of RO water (or 132 mMacetate buffer pH 5) as mobile phase through each membrane wasdetermined. Membranes were presoaked in testing fluid for at least 10minutes prior to testing, flushed with ˜300 mL of testing liquid, thenthe amount of the testing liquid that passes under 100 kPa appliedpressure through a circular membrane coupon of 7.7 cm diameter (withactual 7.3 cm available diameter) was determined. The flux is expressedin the amount of liquid per surface area per time (kg/m² h).

Porous Structure Imaging:

To probe the gel structure and porosity, environmental scanning electronmicroscopy (ESEM) was used to image the membrane in the wet state. Asmall coupon (˜7×5 mm) was wetted by soaking in distilled water for10-15 minutes then examined using an ESEM instrument (FEI Quanta FEG 250ESEM). The sample was placed on cooling stage to adjust the temperatureto 5° C., and the image was examined at low pressure level (4.5-5.5torr) and 50-55% relative humidity.

To probe the membrane structure in the dry state, Tescan Vega II LSUscanning electron microscope (SEM) (Tescan, Pa., USA) was used to imagegold-coated membranes with voltage set to 10-20 kV.

Pore Size Measurements:

Membrane pore size (diameter) was measured using a CFP-1500-AE CapillaryFlow Porometer (Porous Materials Inc., Ithaca, N.Y.), operated by CapWinsoftware (V.6).

A small disc of membrane (2.5-cm diameter) was soaked in Galwick wettingliquid (Porous Materials Inc., surface tension=15.9 dynes/cm) for 10min, then it was gently squeezed between two pre-wetted filter paperdiscs (Whatman 5-70 mm) to remove excess solution, and the thickness ofthe wetted membrane was determined using a micrometer. The membrane discwas then placed on a 2.5-cm stainless steel mesh support disc. Thesupport disc loaded with the test membrane was placed in the designatedholder, with the membrane facing up. The metal cover was then gentlyplaced on the holder and the test was run within the pressure range of0-200 psi.

Coupling Protocol for Conjugating Protein-A Ligand to Click AlkeneMembrane:

To examine the feasibility of chemically binding biomolecules (withthiol functionality) to alkene membranes via a hydrothiolation clickreaction, an engineered protein A ligand containing a cysteine residuewas coupled to alkene membrane(s) (of different chemical formulas) andthe bioactivity of the immobilized ligand was examined.

Protein A ligand lyophilized powder (r-Protein A-cys) was dissolved inPBS (20 mM sodium phosphate, 0.15 M NaCl, pH 7.4) to make a stocksolution of 50 mg/mL. To make a coupling solution for each membrane, 0.4mL of ligand stock solution was transferred into a small ziplock plasticbag (5×8 cm), to which 1.6 mL of 2 M phosphate buffer (pH 7.2) was addedand then 50 μL of initiator (4,4′-azobis(4-cyanovaleric acid), ACVA) inDMAc (150 mg/mL) was added. The reaction solution was mixed well. Thefinal reaction solution had a volume of ˜2.0 mL, and contained about 20mg of ligand, and about 7.5 mg of initiator.

Alternatively, ACVA was dissolved in the reaction buffer (2 M phosphate,pH 7.2) at a concentration of 5 mg/mL in order to avoid using DMAc. Forlow salt experiment, the initiator was dissolved in 0.5 M phosphate at aconcentration of 7.5 mg/mL.

To the bag loaded with coupling reactants, a 4×7-cm membrane coupon(pre-wetted in water) was added. The bag was shaken for a minute, thenirradiated with UV light (˜365 nm) for 10 minutes. After irradiation wascomplete, the coupling solution was decanted, then 15-20 mL of washingbuffer solution (0.1 M phosphate, pH 7.2) was added and the membrane wasplaced on the shaker for 10-15 minutes. The washing cycle was repeatedthree times, after which the membrane was either: (i) transferred into 8mL of trehalose solution (10 wt. %), shaken for 10-15 minutes, and driedin an oven (50° C.) for 20-30 min; or (ii) stored in 0.1 M phosphatebuffer.

For coupling in the presence of additives, ACVA was dissolved in 0.5 Mpotassium phosphate (pH 7.2) to make a solution having a concentrationof 7.5 mg/mL. Protein A ligand was dissolved in 20 mM sodium phosphatebuffer (pH 7.2) to make a 50 mg/mL stock solution. In each of threesmall bags (5×8 cm), 0.25 mL of ligand stock solution was mixed with0.25 mL of initiator solution and 50 μL of an additive were added(cysteamine-HCl to reaction B bag, and 1-mercaptoethanol to reaction Cbag).

After mixing the reaction solutions well, a 25-mm diameter membrane discwas placed in each bag and the reaction bags were shaken well, thenirradiated by UV light for 10 minutes. The reaction solution wasdecanted, then membrane coupons were washed three times using 0.1 Msodium phosphate buffer (pH 7.2) and shaken for 10-15 minutes. Thecomposite membrane coupons were stored in buffer (0.1 M sodiumphosphate, pH 7.2) and tested for bio-affinity to IgG protein, asoutlined previously.

Protein a Ligand Density on Composite Membranes:

To measure the Protein A ligand density on the coupled membrane, theamount of the uncoupled protein, which remained after the couplingreaction, was determined and subtracted from the total ligand amount togive the amount of the coupled ligand, then it was divided by themembrane volume (mL) to express density in mg ligand per mL of membrane.

To determine the Protein A amount in solution, a series of proteinsolutions in 0.1 M phosphate buffer (pH 7.2) were made, the absorbanceat 280 nm was measured for each, and a calibration curve was constructedfrom which the slope was determined.

For selected membrane formulas, coupons of 4 cm×7 cm were cut and theirthicknesses were measured, from which the volume was calculated. Thecoupling reaction was carried out as outlined previously, and 20 mg wereloaded to each membrane coupling reaction, individually. When the UVreaction was complete, the reaction solution was collected in a tube,then 3-5 mL of 0.1 M phosphate buffer were added to the reaction bag andused to wash the membrane by shaking for 20-25 min, then the resultingsolution was added to the collection tube.

The washing cycle was repeated two additional times, then the finalsolution absorbance was measured and the amount of uncoupled protein wascalculated using the calibration curve slope. The coupled ligand amountwas determined by taking the difference between the total reacted anduncoupled amounts.

Post-Polymerization Chemical Modification with Carboxylate Groups:

Additional membranes were synthesized and then modified by exploitingthe click reaction to graft 1-mercaptosuccinic acid with alkenemembranes in order to introduce carboxylate groups to the polymerbackbone.

For example, a coupon having a diameter of 7.7 cm was cut from amembrane comprising a plurality of alkene functional groups and the fluxwas measured (initial flux). The coupon was then transferred into aplastic bag.

For these membranes, the grafting reaction was carried out in aqueousconditions. Mercaptosuccinic acid was dissolved in 6 mL deionized water,then 0.3 mL of ACVA initiator solution (150 mg/mL in DMAc) were added.The reaction solution was mixed well then added into the bag and mixedwith the membrane coupon. This mixture was then irradiated in a UVchamber (approx. 350 nm) for 10 minutes.

After UV light exposure, the membrane coupons were removed from the bagand each coupon was rinsed twice with 20 mL of water, then twice with 20mL of 0.1 M NaOH solution, and finally rinsed twice with 20 mL of water.The RO water flux and acetate buffer solution flux of the membrane weredetermined, then a small disc (25-mm diameter) was cut and used todetermine cation exchange (CEX) IgG binding capacity.

Post-Polymerization Chemical Modification with Hydrophobic Ligands toGenerate Hydrophobic Interaction Chromatography (HIC) Membranes

Representative alkene-containing membranes were made as outlined above,then small coupons (4×7 cm each) were individually placed in smallplastic zip-bags, each loaded with 3 mL of dimethylacetamide (DMAc) thatcontained 120 mg of 1-octanethiol and 10 mg of photoinitiator (ACVA).The reaction bags (with membrane coupons) were transferred into a closedUV chamber and irradiated with UV light for 10 minutes. Membranes wererinsed twice with 10 mL of DMAc, then rinsed once with 10 mL of 30%isopropanol in water, then rinsed twice with 10 mL of water. Membranecoupons were removed and dried in the oven (40° C.) for 10-15 minutes.

Binding Capacity Measurement:

Bio-Affinity IgG Binding Capacity

A 25-mm diameter membrane disc was placed in a 25-mm Natrix-StainlessSteel (SS) holder. 20 mL of binding buffer (20 mM sodium phosphate, 150mM NaCl, pH 7.4) was passed through to equilibrate (˜160-200 bedvolume/min). In the binding step, 0.5 mg/mL polyclonal IgG in bindingbuffer was passed through at flow rate of 1 mL/min until the UVabsorbance of the effluent exceeded 10% of the feeding solution, andthen 10-15 mL of buffer was passed through to remove unbound protein atflow rate 2 mL/min. In the elution step, the bound IgG was eluted bypassing 10-14 mL of elution buffer (0.1 M glycine-HCl, or 0.1 M sodiumcitrate, both at pH 3) at flow rate 2 mL/min.

Cation Exchange IgG Binding Capacity

A 25-mm membrane disc was placed in a 25-mm Natrix-SS holder and 20 mLof binding buffer (132 mM sodium acetate, pH 5.0) were passed through toachieve equilibration. Then protein solution (0.5 mg/mL human polyclonalIgG (Equitech-Bio Inc.) in binding buffer) was passed through until theUV absorbance of the effluent exceeded 10% of the feeding solution, andthen 10-15 mL of buffer was passed through the cell to wash unboundprotein. In the elution step, the bound IgG was eluted by passing 10 mLof elution buffer (132 mM sodium acetate, 1 M NaCl, pH 5.0; or 50 mMTris, 0.5 M NaCl, pH 8.5).

Hydrophobic Interaction Mode IgG Binding Capacity

A 25-mm membrane disc was placed in a 25-mm Natrix-SS holder and 20 mLof binding buffer (50 mM sodium phosphate, 1 M ammonium sulfate, pH 6.5)was passed through to achieve equilibration. Then, a protein solution(0.5 mg/mL human polyclonal IgG (Equitech-Bio Inc.) in binding buffer)was passed through until the UV absorbance of the effluent exceeded 10%of the feeding solution. Subsequently, 15-20 mL of buffer was passedthrough the cell to wash unbound protein. In elution step, the bound IgGwas eluted by passing 10 mL of elution buffer (50 mM sodium phosphate,pH 7.0).

Post-Polymerization Chemical Modification with Carboxylate Groups:

Selected membranes were modified by exploiting the click reaction tograft 1-mercaptosuccinic acid with alkene membranes in order tointroduce carboxylate groups to the polymer backbone. For each membrane,a coupon having a diameter of 7.7 cm was cut and the flux was measured(initial flux). The coupon was then transferred into a plastic bag.

For modification in N,N′-dimethylacetamide (DMAc), a reaction solutionwas made by dissolving 0.3 g of 1-mercaptosuccinic acid in 6 mL of DMAc.Then, 0.3 mL of ACVA initiator solution (150 mg/mL in DMAc) were addedto the thiol solution. Finally, the complete reaction solution was addedto the reaction bag containing the membrane.

For aqueous reaction conditions, 6 mL deionized water were used insteadof DMAc to dissolve mercaptosuccinic acid and 0.3 mL of ACVA initiatorsolution (150 mg/mL in DMAc) were added to it.

The bag was shaken well to ensure complete impregnation of the membranewith reaction solution, then it was irradiated by light (˜365 nm) for 10minutes, after which the reaction solution was discarded. 20 mL of waterwere added to the bag and the membrane was washed with agitation for 10minutes. The wash solution was discarded and another 20 mL of water wereadded and the cycle was repeated. The membrane was washed twice morewith 0.1 M NaOH (20 mL, 10 min. each). Finally, the membrane was washedtwice more with water, and the flux of the membrane was determined(after flux).

Example 1—Formulation with TEGDV Co-Monomer

In certain embodiments, a hydrophilic co-monomer is included to helptune the membrane permeability.

In this membrane formulation class, 2,2′-(ethylenedioxy)diethanethiol(EDDET) monomer, and tri(ethylene glycol) divinyl ether (TEGDV), asco-monomer, were used as building monomers and1,3,5-triallyl-1,3,5-triazine-2,4,6(1H,3H,5H)-trione (TATATO) was usedas a crosslinker. The solvent system included N,N′-dimethylacetamide(DMAc), (±)-1,3-butanediol (Budiol), di(propylene glycol)methyl etheracetate (DPMA), and water in variant amounts.4-(2-Hydroxyethoxy)phenyl-(2-hydroxy-2-propyl)ketone (IRGACURE 2959) wasused as photoinitiator to start the polymerization radical reaction.

Reaction mixtures based on these ingredients were formulated accordingto the tabulated data and all components were added and mixed wellexcept the dithiol which was added 10-15 min prior to casting (to avoidany premature polymerization initiated by ambient light). Membranes werecast and polymerized as described previously. Mass gain and wetting timewere determined and the initial flux of each membrane's coupon (7.7 cmin diameter) was measured using RO water.

The results (as shown in FIG. 1) show that it is possible to makemembranes of various alkene/thiol ratio and versatile permeability, asindicated by water flux. The results also show that the solvent systemmay be used to help tune the membrane porosity and, as a result, themembrane permeability. For example, increasing 1,3-butanediol contentwhile decreasing di(propylene glycol)methyl ether acetate (DPMA) contentin the formula increased the membrane flux (Formulas CLK-EN-12 vs.CLK-EN-17 and CLK-EN-90 vs. CLK-EN-81). The results also show thatreplacing the crosslinker (TATATO) with the divinyl triethylene glycolextender has decreased membrane flux (Formulas CLK-EN-12 vs. CLK-EN-16).

While not wishing to be bound by any particular theory, 1,3-butanediolmay be considered a non-solvent to the polymeric chain, and thereforetends to increase gel porosity during the polymerization step. As aresult, permeability (expressed in water flux) increases. DPMA may playthe same role.

Example 2—Formulation with TEGDV Co-Monomer—Effect of InitiatorConcentration

In this example, the effects of the concentration of photoinitiator onthe polymerization process and the resultant membrane properties wereexamined. Similar to the previous class,2,2′-(ethylenedioxy)diethanethiol (EDDET) monomer, and tri(ethyleneglycol) divinyl ether (TEGDV) co-monomer, and1,3,5-triallyl-1,3,5-triazine-2,4,6(1H,3H,5H)-trione (TATATO)crosslinker were used. The solvent system includedN,N′-dimethylacetamide (DMAc), butanediol (Budiol), di(propyleneglycol)methyl ether acetate (DPMA), and water in varying quantities.

The amount of the 4-(2-hydroxyethoxy)phenyl-(2-hydroxy-2-propyl)ketone(IRGACURE 2959) photoinitiator was varied, according to FIG. 2 for twosets of polymerization reactions, one at 10 minutes polymerization time(CLK-EN-27 and CLK-EN-45) and the other (CLK-EN-137 to 140) at 6 minutespolymerization time. The shorter polymerization time was examined toallow a better discrimination, based on initiator amount, of theresultant membrane properties because the hydrothiolation click reactionis a fast reaction.

The results, as shown in FIG. 2, suggest that initiator amount has aneffect on membrane permeability. The flux tends to decrease as theinitiator amount increases. This effect demonstrated itself at both 10and 6 minutes polymerization time. While not wishing to be bound by anyparticular theory, more initiator means that the polymerization proceedsto a higher rate of conversion; the likely result is a denser polymericnetwork.

Example 3—Formulation with TEGDV and DATA as Co-Monomers

In this example, another hydrophilic co-monomer (N,N′-diallyltartramide(DATA)) was examined. While not wishing to be bound by any particulartheory, the two hydroxyl groups in this molecule increase theamphiphilic nature of the polymer, which may enhance phase separation asthe polymer chains grow, thereby improving the porosity of the finalgel.

In this membrane formulation class, 2,2′-(ethylenedioxy)diethanethiol(EDDET) monomer and tri(ethylene glycol) divinyl ether (TEGDV) and(+)-N,N′-diallyltartramide (DATA) co-monomers, were used as buildingmonomers with 1,3,5-triallyl-1,3,5-triazine-2,4,6(1H,3H,5H)-trione(TATATO) as a crosslinker.

The solvent system included N,N′-dimethylacetamide (DMAc),(±)-1,3-butane diol (budiol), di(propylene glycol)methyl ether acetate(DPMA), and water in varying amounts.4-(2-Hydroxyethoxy)phenyl-(2-hydroxy-2-propyl)ketone (IRGACURE 2959) wasused as photoinitiator to start the polymerization reaction.

The reaction components were mixed well together, except the dithiolwhich was added 10-15 min prior to casting. The membranes were cast andpolymerized as described previously. Mass gain and wetting time weredetermined, then the initial flux of each membrane coupon (7.7 cm indiameter) was measured using RO water.

The results for this example demonstrate again the effect of the solventsystem on the membrane permeability. As seen when comparing formulaCLK-EN-99 with CLK-EN-104 (FIG. 3), reducing the amount of 1,3-butandioland increasing the amount of N,N′-dimethylacetamide (DMAc) result in aremarkable reduction of permeability (water flux decreased from about10,000 to about 1700 kg/m² h).

In general, 1,3-butanediol and water are considered non-solvents or poorsolvents to the polymeric chain; therefore porosity of membranes formedin these solvents is increased. On the contrary, DMAc is considered agood solvent that helps solvate the polymeric chain as it forms; as aresult, porosity and permeability are reduced.

Example 4—Formulation with DATA as Co-Monomer

In this example, the use of N,N′-diallyltartramide (DATA) as the soleco-monomer was examined. DATA molecules have internal amide bonds (whichTEGDV molecules do not have); these may add some mechanical strength tothe resulting membrane.

In this membrane formulation class, 2,2′-(ethylenedioxy)diethanethiol(EDDET) monomer, (+)-N,N′-diallyltartramide (DATA) as co-monomer, and1,3,5-triallyl-1,3,5-triazine-2,4,6(1H,3H,5H)-trione (TATATO) as acrosslinker were used to make the membranes. The solvent system includedN,N′-dimethylacetamide (DMAc), (±)-1,3-butanediol (Budiol), di(propyleneglycol)methyl ether acetate (DPMA), and water in varying amounts.4-(2-Hydroxyethoxy)phenyl-(2-hydroxy-2-propyl)ketone (IRGACURE 2959) wasused as the photoinitiator.

The reaction components (FIG. 4) were mixed well together, except thedithiol which was added 10-15 min prior to casting. The membranes werecast and polymerized as described previously. Mass gain and wetting timeof the dried membranes were determined, then the initial flux of eachmembrane's coupon (7.7 cm in diameter) was measured using R.O. water.

The results (FIG. 4) show that increasing DATA content (from 3.3% to7.6%), with concomitant decrease in the crosslinker content (from 16.3%to 9.9%), reduced the membrane flux.

Reducing dithiol (EDDET) content in this system lead to an increase inthe membrane flux (CLK-EN-149 to −151). While not wishing to be bound byany particular theory, it is possible that higher dithiol content helpsin connecting smaller growing polymeric chains, resulting in denser gelwith higher mass gain and lower flux.

These membranes were examined by environmental scanning electronmicroscopy (ESEM), which showed porosity of the gel (FIG. 6).

Example 5—Formulation with Dialkyne Crosslinker

In this example, dialkyne molecule (1,7-octadiyne) was examined as anadditional crosslinker that can boost the unsaturated carbon-carbon bondpopulation within the formulated membrane. This can be beneficial as itincreases the possibility of engrafting the gel with thiol functional(bio)molecules.

In this membrane formulation class, 2,2′-(ethylenedioxy)diethanethiol(EDDET) monomer and tri(ethylene glycol) divinyl ether (TEGDV)co-monomer were used as building monomers, while1,3,5-triallyl-1,3,5-triazine-2,4,6(1H,3H,5H)-trione (TATATO) and1,7-octadiyne (OctDi) were used as crosslinkers. The solvent systemincluded N,N′-dimethylacetamide (DMAc), (±)-1,3-butanediol (Budiol),di(propylene glycol)methyl ether acetate (DPMA), and water, all invarying amounts. 4-(2-Hydroxyethoxy)phenyl-(2-hydroxy-2-propyl)ketone(IRGACURE 2959) was used as photoinitiator.

The reaction components (FIG. 7), except the dithiol (EDDET), were mixeduntil all dissolved. Then, EDDET was added 10-15 min prior to casting.Membranes were cast and polymerized as described previously. Mass gain,wetting time, and initial flux of each membrane coupon (7.7 cm indiameter—using R.O. water) were determined.

Results shown in FIG. 7 demonstrate that it is possible to formulatemembranes with octadiyne as a co-crosslinker. Decreasing the amount ofcrosslinkers (TATATO & OctDi) results in a composite membrane havinglower permeability. ESEM shows the porous structure of these membranes(FIG. 8).

Example 6—Post-Polymerization Grafting of Alkene/yne Membrane withCarboxylate Groups by Hydrothiolation

Carboxylate groups are hydrophilic in nature and known to increase gelswelling due to the strong hydration of the ionized form (e.g.,polymethacrylates). Increasing the carboxylate group content in the gelincreases the gel swelling, which in turn decreases the flux. On theother hand, carboxylic acid groups (i.e., acid form) are consideredrelatively hydrophobic; a gel having carboxylic acid moieties in theirnon-ionized form does not swell as much, and the flux increases. Thisphenomenon is known as hydrogel pH sensitivity.

To demonstrate the capability of grafting a click alkene polymer withnew functionality by using a post-polymerization click reaction,alkene/yne membranes having high water flux were made (FIG. 9). Thesemembranes were subject to click hydrothiolation functionalization with athiol-acid molecule. The permeability of the modified membrane,expressed in flux, was determined and compared with the initial fluxprior to the grafting reaction.

The post-polymerization grafting click reaction was carried out in DMAcor water. The flux of each modified membrane was measured using ROwater. To probe the pH sensitivity of the modified membranes, acetatebuffer (132 mM acetate, pH 5) was used.

The flux of the modified membranes decreased as compared to their fluxbefore grafting (Table 1). When the flux was determined using acetatebuffer at pH 5, the flux increased because the carboxylate group wasconverted into the hydrophobic form (carboxylic acid), which decreasedthe gel swelling. Furthermore, when the membrane was flushed with 0.1 MNaOH solution, the R.O. flux dropped which confirms the membrane pHsensitivity because of the deprotonation of the carboxylic acid groupsto form carboxylate groups at basic pHs. See also FIG. 10.

TABLE 1 Membrane flux (kg/m²h) in solvents with varying pH RO NaOHInitial Flux pH 5 Acetate RO Flux Alkene/ RO After RO Flux After Afterthiol Formula Flux Grafting Grafting Grafting ratio CLK-EN-015 219173806 21333 3318 1.35 CLK-EN-042 27143 6138 22399 5405 1.44 CLK-EN-04623509 14676 21896 13650 1.44

To examine the effect that the reaction medium has on the properties ofthe modified membrane, the grafting hydrothiolation reaction was carriedout on a membrane in DMAc and separately in water, and the flux of themodified membranes in different media was evaluated. As shown below(Table 2), membranes grafted in water have lower flux than counterpartmembranes that were modified in DMAc. This coincides with generalunderstanding that the hydrothiolation reaction is more reactive inpolar solvents, and in aqueous media in particular. See also FIG. 11.

TABLE 2 Effect of solvent during grafting reaction on membrane flux(kg/m²h) RO Flux after RO Flux after Initial modification modificationin Alkene/thiol Formula RO Flux in H₂O DMAc ratio EN-112 15758 92 31411.23 EN-113 13612 0 833 1.21 EN-133 15531 5980 13103 1.08

Example 7—Post-Polymerization Grafting of Alkene/yne Membrane withProtein A by Hydrothiolation

To demonstrate that the click alkene membranes may be grafted with abiological ligand, protein A having with cysteine terminal functionality(rProtein A-cys) was coupled to selected click alkene membranes asdescribed above. The binding capacity of the membrane with graftedprotein A moieties was determined by examining its bio-affinity for IgGprotein, following the binding/elution protocol, as outlined above.

Binding capacity results (Table 3) demonstrate that the proteinA-modified membranes are capable of binding IgG. This would not bepossible if the ligand was not coupled to the gel membrane or wasinactive. The elution solution for EN-151 was citrate buffer solution(0.1 M, pH 3), while glycine hydrochloride buffer solution (0.1 M, pH 3)was used in the elution step for EN-134 and EN-152.

It is interesting to note that the coupling reaction took place evenwhen the alkene-to-thiol ratio was less than 1, which suggests that theoriginal polymerization reaction did not consume all alkene groups.Because polymeric chain growth can impede chain mobility during thefinal stages of the polymerization reaction, this is not unexpected. So,it is possible that any excess thiol is coupling to the residual alkenegroups in the polymer.

The results also show that the alkene-to-thiol ratio is not the solefactor in controlling the coupling reaction. For example, a higher ratiodid not result in higher coupling and concomitant bioactivity. Otherfactors such as porosity, surface area, and surfacehydrophilic/hydrophobic nature, contribute to the accessibility of thealkene groups, thereby affecting the coupling reaction.

TABLE 3 IgG binding capacity of engrafted click-protein A membrane IgGBinding Alkene/thiol RO Flux Capacity_(10%B.T) Formula ratio (kg/m²h)(mg/mL) CLK-EN-016 1.363 2189 1.4 CLK-EN-027 1.238 1468 1.3 CLK-EN-1341.074 6979 3.7 CLK-EN-118 1.278 11509 1.6 CLK-EN-124 1.273 14895 2.8CLK-EN-143 0.963 9007 4.9 CLK-EN-149 0.963 3804 5.0 CLK-EN-150 1.0156700 5.5 CLK-EN-151 1.052 7808 6.2 CLK-EN-152 0.963 7674 5.6

Example 8—Effect of Grafting Reaction Time and Amount of Ligand onProperties of Modified Membranes

In order to use the radical hydrothiolization (thiol-ene) reaction tograft protein A to alkene membranes, the reaction must be initiated byUV radiation. Therefore, it was necessary to investigate the effects ofthis exposure on the grafted ligand bioactivity.

One membrane formula was subject to protein A coupling experimentsduring which the light dose (at 365 nm), gauged by exposure time, wasvaried and the effect on bioactivity (as reflected by binding capacity)was examined (Table 4).

The results suggest that varying the exposure time from 10 to 20 minutesdoes not affect the binding capacity of the final modified membrane,regardless of the ligand amount or concentration in the reactionsolution. The results also show that increasing the amount of proteinimproved bioactivity.

TABLE 4 Light and ligand amount effects on coupling reaction TotalReaction ligand Exposure Binding Volume Conc. amount time CapacityExperiment (mL) (mg/mL) (mg) (min) (mg/mL) A 1 10 10 10 0.7 B 1 10 10 200.7 C 2 5 10 10 0.9 D 2 5 10 20 0.8 E 2 10 20 10 1.3 F 2 10 20 20 1.3

Example 9—Effect of the Presence of Competing Additives on Properties ofModified Membranes

To demonstrate that a hydrothiolation reaction is responsible forattaching the cys-protein A ligand to the alkene membrane, the couplingreaction was carried out on small 25-mm diameter discs of the samemembrane (CLK-EN-143) in the presence and absence ofthiol-functionalized molecules, which can compete with the ligand forthe available alkene groups and, therefore, limit the extent of theligand coupling reaction. Indeed, the ligand coupling reaction in thepresence of competing thiol molecules resulted in membranes havingreduced bioactivity, compared to the modified membrane formed in theabsence of thiol-functionalized additives. FIG. 12.

Example 10—Formulation with DATA as Co-Monomer

In this example, the use of N,N′-diallyltartramide (DATA) as the soleco-monomer was examined. DATA molecules have internal amide bonds (whichTEGDV molecules do not have); these may add some mechanical strength tothe resulting membrane.

In this membrane formulation class, 2,2′-(ethylenedioxy)diethanethiol(EDDET) monomer, (+)-N,N′-diallyltartramide (DATA) as co-monomer, and1,3,5-triallyl-1,3,5-triazine-2,4,6(1H,3H,5H)-trione (TATATO) as acrosslinker were used to make the membranes. The solvent system includedN,N′-dimethylacetamide (DMAc), (±)-1,3-butanediol (Budiol), di(propyleneglycol)methyl ether acetate (DPMA), and water in varying amounts, orN,N′-dimethylacetamide (DMAc), (±)-1,3-butanediol (Budiol), di(ethyleneglycol), tri(ethylene glycol), and water in varying amounts.4-(2-Hydroxyethoxy)phenyl-(2-hydroxy-2-propyl)ketone (IRGACURE 2959) wasused as the photoinitiator.

The reaction components (FIG. 4B and FIG. 13) were mixed well together,except the dithiol which was added 10-15 min prior to casting. Themembranes were cast and polymerized as described previously. Mass gainand wetting time of the dried membranes were determined, then theinitial flux of each membrane's coupon (7.7 cm in diameter) was measuredusing RO water.

The results (FIG. 4B and FIG. 13) show that it is possible to feed DATAco-monomer over a wide range by using different solvent systems toobtain a wide range of membrane permeabilities, as indicated by membranewater flux measurements. When these membranes were grafted with ProteinA ligand, they showed a corresponding range of IgG binding capacities(see post polymerization grafting with Protein A section).

These membranes were examined by scanning electron microscopy (SEM),which revealed uniform, highly interconnected porous networks (FIG. 14).

Example 11—Formulation with Tetrathiol Molecule as a Co-Crosslinker

In certain embodiments, pentaerythritol tetrakis(3-mercaptopropionate)(PETM) was used as an additional crosslinker in order to modify theresulting membrane structure and permeability.

In this membrane formulation series, 2,2′-(ethylenedioxy)diethanethiol(EDDET) monomer, and (+)-N,N′-diallyltartramide (DATA) co-monomers, wereused as monomers and1,3,5-triallyl-1,3,5-triazine-2,4,6(1H,3H,5H)-trione (TATATO) andpentaerythritol tetrakis(3-mercaptopropionate) (PETM) were used as anadditional crosslinker. The solvent system included N,N′-dimethylacetamide (DMAc), (±)-1,3-butanediol (Budiol), hexylene glycol, ethyleneglycol (EG), tetra(ethylene glycol) (TetEG) and water in variantamounts. 4-(2-Hydroxyethoxy)phenyl-(2-hydroxy-2-propyl)ketone (IRGACURE2959) was used as photoinitiator to start the polymerization radicalreaction.

Reaction mixtures based on these ingredients were formulated (FIG. 8)and all components were added and mixed well except the dithiol whichwas added 10-15 min prior to casting (to avoid any prematurepolymerization initiated by ambient light). Membranes were casted andpolymerized as described previously. Mass gain and wetting time weredetermined and the initial flux of each membrane's coupon (7.7 cm indiameter) was measured using RO water.

The results (as shown in FIG. 15) show that it is possible to usemulti-arm thiol (PETM) as a second crosslinker to produce membranes ofvarious alkene/thiol ratio with variable permeability, as indicated bywater flux. It is possible to increase the permeability by increasingthe overall crosslinkers content in the gel, as demonstrated in Table 5.SEM shows the porous structure of a representative membrane formulation(FIG. 16).

TABLE 5 Effect of PETM crosslinker on membrane permeability Totalcrosslinker mole % (mol %) mole % RO Flux Formula EDDET PETM TATATO DATAPETM + TATATO (kg/m²h) CLK-EN-314 45.62 7.26 23.94 22.78 31.2 6781CLK-EN-317 45.22 7.07 26.17 21.14 33.24 7995 CLK-EN-323 40.68 12.1625.50 21.42 37.66 14788 CLK-EN-325 40.81 12.28 25.75 20.83 38.02 11812

Example 12—Additional Formulations with Dialkyne Crosslinker

In this set of additional examples, a dialkyne molecule (1,7-octadiyne)was included in the formula as an additional crosslinker to increase theunsaturated carbon-carbon bond population within the polymerizedmembrane.

In this membrane formulation class, 2,2′-(ethylenedioxy)diethanethiol(EDDET) monomer and (+)-N,N′-diallyltartramide (DATA) co-monomers, wereused as monomers and1,3,5-triallyl-1,3,5-triazine-2,4,6(1H,3H,5H)-trione (TATATO) and1,7-octadiyne (OctDi) were used as crosslinkers. The solvent systemincluded N,N′-dimethylacetamide (DMAc), sodium dodecyl sulfate (SDS),ethylene glycol (EG), tetra(ethylene glycol) (TetEG), and water, all invarying amounts. 4-(2-Hydroxyethoxy)phenyl-(2-hydroxy-2-propyl)ketone(IRGACURE 2959) was used as photoinitiator.

The reaction components (FIG. 17), except the dithiol (EDDET), weremixed in the solvent mixture until completely dissolved. Then, EDDET wasadded 10-15 min prior to casting. Membranes were casted and polymerizedas described previously. Mass gain, wetting time, and initial flux ofeach membrane coupon (7.7 cm in diameter—using R.O. water) weredetermined.

Results shown in FIG. 17 demonstrate that it is possible to formulatemembranes with octadiyne as a co-crosslinker that vary widely in theirpermeability. Scanning electron microscopy (SEM) image of arepresentative membrane (CLK-EN-361) shows a uniform, interconnectedporous structure containing small pores (FIG. 18).

Example 13—Cation Exchange IgG Binding Using Membranes Functionalizedwith Carboxylate Groups

Carboxylate groups are hydrophilic in nature and known to increasepolymer swelling due to the strong hydration of the ionized form (e.g.,polymethacrylates). Increasing the carboxylate group content in theporous polymer increases the swelling, which in turn decreases the flux.See Example 6.

The cation exchange binding capacity for protein IgG (which has netpositive charge at pH 5) for each grafted membrane was examined toprovide additional support for the incorporation of the charged cationexchange ligands in the membrane. The membranes' dynamic bindingcapacity for IgG increases with increasing alkene/thiol ratio. See Table6.

TABLE 6 Effect of attaching carboxylate groups on membrane flux (kg/m²h)and IgG binding capacity RO Flux Acetate pH 5 CEX IgG Initial RO AfterFlux After Binding Flux Grafting Grafting Capacity_(10%B.T) Alkene/thiolFormula (kg/m²h) (kg/m²h) (kg/m²h) (mg/mL) ratio CLK-EN-237 15432 172513353 19.5 1.101 CLK-EN-227 13664 705 11848 22.2 1.101 CLK-EN-224 17189773 14375 28.1 1.129 CLK-EN-287 6972 20 5198 37.5 1.135 CLK-EN-235 1224334 10041 37.3 1.142 CLK-EN-291 10172 24 7539 37.9 1.159 CLK-EN-298 69180 5533 44.2 1.159 CLK-EN-301 13841 15 10684 54.7 1.211 CLK-EN-256 162467 15777 50.6 1.222

Example 14—Post-Polymerization Grafting of Alkene/yne Membrane (Madewith DATA Co-Monomer) with Protein A by Hydrothiolation

To demonstrate that the click alkene membranes may be grafted with abiological ligand, an engineered Protein A ligand containing aC-terminal cysteine residue (rProtein A-cys) was coupled to selectedclick alkene membranes that were made as described above. The IgGbinding capacity of the membrane with grafted Protein A ligand wasdetermined, as outlined above.

IgG binding capacity results (Table 7) of the membrane with higher DATAcontent (i.e., >8 wt. % in the polymerization mixture) demonstrate thatthe Protein A-modified membranes are capable of binding more IgG thanmembranes having a lower relative concentration of DATA monomer (i.e.,<8 wt. % in the polymerization mixture).

The results also suggest that the alkene-to-thiol ratio is not the onlyvariable correlated to membrane IgG binding capacity performance. Otherfactors such as porosity, surface area, and surfacehydrophilic/hydrophobic nature, likely also play important roles, asthey contribute to the accessibility of the alkene groups, therebyaffecting the Protein A ligand coupling reaction.

TABLE 7 IgG binding capacity of Protein A-grafted membranes (withincreased DATA co-monomer in reaction solution) Ligand RO density onAlkene/ Flux IgG Binding ProA wt. % thiol (kg/ Capacity_(10%B.T)membrane Formula DATA ratio m²h) (mg/mL) (mg/mL) CLK-EN-301 8.26 1.21113841 9.6 6.9 CLK-EN-227 8.47 1.101 13664 7.3 5.9 CLK-EN-237 8.48 1.10115432 8.3 CLK-EN-256 8.52 1.222 16246 9.2 6.4 CLK-EN-298 8.52 1.159 691810 5.7 CLK-EN-291 8.54 1.159 10172 9.8 7.0 CLK-EN-235 8.67 1.142 1224310.8 CLK-EN-287 8.96 1.135 6972 10.2 CLK-EN-224 9.10 1.129 17189 6 5.3

Example 15—Post-Polymerization Grafting of Alkene/yne Membrane (Madewith PETM Co-Crosslinker) with Protein A by Hydrothiolation

In this class of membrane, the tetrathiol crosslinker PETM was used asan additional crosslinker to provide another tool to tune the degree ofcrosslinking and permeability of the membranes. The presence and surfacedensity of post-polymerization alkene functional groups were probed byfirst grafting Protein A ligand to these membranes using the radicalhydrothiolation (thiol-ene) reaction. Then the IgG binding capacity ofthe Protein A-grafted membranes was assessed as described previously.

As shown below (Table 8), the results suggest that membranes made withthis co-crosslinker possess residual alkene groups that are functionaland accessible for the rProtein A-cys hydrothiolation (thiol-ene)reaction on the membrane surface.

TABLE 8 IgG binding capacity of Protein A-grafted membranes (made withPETM co-crosslinker) IgG Binding Alkene/thiol RO Flux Capacity_(10%B.T)Formula ratio (kg/m²h) (mg/mL) CLK-EN-314 1.128 6781 10.3 CLK-EN-3171.176 7995 10.7 CLK-EN-323 1.183 14788 8.9 CLK-EN-325 1.174 11812 11.8

Example 16—Post-Polymerization Grafting of Alkene/yne Membrane withHydrophobic Ligand to Generate Hydrophobic Interaction Chromatography(HIC) Media

A hydrophobic thiol-terminated molecule was grafted to membranescontaining residual alkene functional groups using a photoinitiatedclick (thiol-ene) hydrothiolation reaction. Three membranes (EN-224,EN-291, and EN-301) were prepared (as outlined previously in theexperimental methods section) for use in subsequent grafting reactions,namely to introduce 1-octanethiol onto the membrane, as described in thegeneral methods section.

Attachment of the hydrophobic thiol to the membranes was expected tosignificantly decrease the surface hydrophilicity of the graftedmembrane versus the ungrafted membrane. Indeed, the wetting timeconsistently increased post-grafting for all of the membraneformulations tested (Table 9).

The hydrophobic ligand-modified membranes were anticipated to serve aseffective hydrophobic interaction chromatography (HIC) media, bindingIgG at high salt concentrations. This phenomenon constitutes the basisof hydrophobic interaction chromatography, a well-known techniqueutilized in bio-separation process for biologicals purification.

Results (Table 9) demonstrate that the modified membrane indeed can bindprotein in high salt conditions.

TABLE 9 Properties of alkene/yne membranes grafted with hydrophobicligand Wetting HIC Binding Alkene/thiol Time Capacity_(10BT%) Formularatio Before After (mg/ml) CLK-EN-224 1.129 1 Sec  6 Sec 10.6 CLK-EN-2911.159 1 Sec 20 Sec 12.4 CLK-EN-301 1.211 1 sec 20 sec 10.8

Example 17—One-Step Polymerization Reaction for Making FunctionalizedHIC-Click Membranes Using Hydrothiolation Reaction

In this example, the flexibility of click chemistry is demonstrated as aclick membrane functionalized with a hydrophobic ligand is demonstrated;a single polymerization step forms the membrane polymer network with ahydrophobic ligand (1-octane thiol) included in its network. Bycontrolling the thiol/alkene ratio in the polymerization mixture, theresidual alkene population can be varied, and, in principal, can be usedin later steps to anchor additional molecules or ligands having the sameor different chemical or physical properties.

In this membrane formulation class, 2,2′-(ethylenedioxy)diethanethiol(EDDET) monomer and both 1,4-dithioerythritol (DDT) and(+)-N,N′-diallyltartramide (DATA) co-monomers, were used as buildingmonomers, while 1,3,5-triallyl-1,3,5-triazine-2,4,6(1H,3H,5H)-trione(TATATO) and 1,7-octadiyne (OctDi) were used as crosslinkers. One-octanethiol was included in the formula to add a hydrophobic pendant or endgroup to the polymer. The solvent system included N,N′-dimethylacetamide(DMAc), sodium dodecyl sulfate (SDS), ethylene glycol (EG),tetra(ethylene glycol) (TetEG), and water, all in varying amounts.4-(2-Hydroxyethoxy)phenyl-(2-hydroxy-2-propyl)ketone (IRGACURE 2959) wasused as photoinitiator.

The reaction components (FIG. 19), except the dithiol (EDDET), weremixed in the solvent mixture until completely dissolved. Then, EDDET wasadded 10-15 min prior to casting. Membranes were cast and polymerized asdescribed previously. Mass gain, wetting time, and initial flux of eachmembrane coupon (7.7 cm in diameter—using R.O. water) were determined.

As shown in FIG. 19, it is possible to make membranes with variablepermeability and IgG binding capacity through a single polymerizationstep that includes a hydrophobic ligand in the reaction mixture. SeeTable 10.

TABLE 10 Functionalized HIC-click membrane use in bio-separation HICMode IgG Alkene/thiol RO water Flux Binding Capacity_(10BT%) Formularatio (kg/m²h) (mg/mL) CLK-EN-411 1.237 4835 7.9 CLK-EN-417 1.093 136358.4

Example 18—Post-Polymerization Step-Wise Grafting Process for Controlledor Extended Graft Architecture Via Hydrothiolation

One major advantage of using hydrothiolation for gel functionalizationis the ability to use the same reaction chemistry to introduce othermonomers in later steps. Moreover, because of the specificity thatcharacterizes click chemistry, it is possible to perform multi-stepgrafting processes, which can introduce pendant building blocks andfunctional groups in a very controlled manner.

To demonstrate this capability, selected membranes containing residualalkene functional groups were subject to stepwise grafting processes.The net result of this two-step grafting process is to build an armextending out of the surface that has defined structure and length, asshown in FIG. 20.

In the first step, a dithiol monomer (EDDET) was used in excess toconvert the gel surface functionality from alkene to thiol groups.Membrane coupons of 7.7-cm diameter were weighed then wetted with water,after which the coupons were transferred individually to plastic zipbags loaded with 4 mL of 10 wt % of dithiol (EDDET) in DMAc and 18 mg ofphotoinitiator (ACVA). Each reaction bag was stirred then exposed to UVlight for 7 minutes, then the coupons were rinsed with 10 mL DMAc.

In the second step, an excess of di-alkene monomer (DATA) was reactedwith the thiol-enriched membrane via a hydrothiolation grafting reactionto make a final membrane that contains alkene functional groups extendedfrom the surface. Membrane coupons were transferred individually to newplastic zip bags loaded with 4 mL of 10 wt % of di-alkene monomer (DATA)in DMAc with ˜18 mg of photoinitiator (ACVA). The reaction bags wereexposed to UV light for another 7 minutes, then the membrane couponswere rinsed with DMAc, followed by several washes in water, then driedat room temperature. The final membrane weight was recorded.

Results, as shown below in Table 11, demonstrate that membrane mass gainincreased slightly while permeability (measured by water flux) decreasedsignificantly subsequent to the two step grafting reaction. Graftedmembrane with permeabilities >1000 kg/m² h were used for subsequentProtein A ligand attachment to help probe for the successfulincorporation of reactive alkene groups via this process and the effecton membrane protein binding capacity. Indeed, measurable IgG bindingcapacity indicates successful ligand grafting to the membrane surface(Table 11). Also, an increase in IgG binding capacity, post-grafting, isonly seen for CLK-EN-224 where the mass gain was the greatest,suggesting the highest grafting yield.

This approach has a strong potential for modifying and optimizing thegraft structure as it provides an efficient tool to construct awell-defined multi-unit grafts (or branches) that extend from thesurface to modify the membrane surface properties, reactive groupdensity, and/or permeability.

TABLE 11 Membrane performance after two-step grafting reaction IgGBinding Mass gain Capacity_(10%B.T) RO Flux wt. % (mg/mL) (kg/m²h)Alkene/thiol After After After Membrane ratio Initial Reaction InitialReaction Initial Reaction CLK-EN-224 1.129 238 251 6 11.1 17189 3996CLK-EN-287 1.135 267 270 10.2 NA 6972 170 CLK-EN-291 1.159 264 266 9.8NA 10172 250 CLK-EN-301 1.211 243 249 9.6  8.7 13841 4151

Example 19—Double Polymerization Process for Constructing CovalentlyConnected “Two Phase” Membranes

Another approach to exploit the capability of alkene-containingmembranes to undergo click hydrothiolation reactions involves performingin situ hydrothiolation polymerization of monomers/crosslinkers thatwill form a second polymeric phase within the pores, and simultaneouslycovalently bond it to the underlying gel.

A membrane formula that has high permeability (CLK-EN-224) was selectedto make four sheets of the base membrane (first phase) on a pre-weighed7″×8″ porous support substrate sheet (non-woven polypropylene mesh), asdescribed above. Each sheet of the CLK-EN-224 membrane was individuallyplaced on polyethylene sheet and impregnated with 12 g of polymerizationsolution described in FIG. 21—A/B/C/D. The impregnated membrane wassubsequently covered with another polyethylene sheet and was pressedgently in a circular motion by hand in order to remove excess solutionand any entrapped air bubbles. The polymerization process was initiatedby irradiating with UV light (˜350 nm) in a closed chamber for 10 min,then washed and dried as described above (General methods—Membranepreparation section).

The final weight and mass gain of each membrane were determined, thenthe increase of mass gain due to the grafted second phase polymerizationwas calculated for each formula. The double polymerization membraneswere then grafted with mercaptosuccinic acid to introduce carboxylategroups to the membrane gel (see general methods section), which allowthe membrane to function as a cation exchange media for proteinbio-separation. In addition, the membranes were also grafted withprotein A ligands, which enable the membrane to function as bio-affinityseparation media (see general methods section for grafting and testingprotocols).

Examining the resultant membranes (FIG. 22, CLK-EN-224 A/B/C/D) fortheir mass gain, permeability (flux), and binding capacity (in bothcation exchange and bio-affinity modes) demonstrates that it is possibleto construct a second phase polymeric gel within the first gel phase,and as a result, make a final composite with unique properties.

Mass gains of the “two-phase” membranes were higher than the basemembrane, and the flux values were lower (FIG. 22). Binding capacitiesof the “two-phase” membranes were different than the base membranes. CEXIgG binding capacities appear to be inversely correlated to mass gainbut directly correlated to water flux.

INCORPORATION BY REFERENCE

All of the U.S. patents and U.S. patent application publications citedherein are hereby incorporated by reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

We claim:
 1. A composite material, comprising: a support member,comprising a plurality of pores extending through the support member;and a macroporous cross-linked gel, wherein the cross-linked gelcomprises a polymer derived from a first monomer and a firstcross-linker; wherein the macroporous cross-linked gel is located in thepores of the support member; the macropores of the macroporouscross-linked gel are smaller than the pores of the support member; thefirst monomer comprises two thiol functional groups; and the firstcross-linker comprises (i) at least three carbon-carbon double bonds,(ii) at least two carbon-carbon triple bonds, or (iii) at least onecarbon-carbon triple bond and at least one carbon-carbon double bond. 2.The composite material of claim 1, wherein the first monomer is2,2′-(ethylenedioxy)diethanethiol (EDDET), 1,2-ethanedithiol,1,4-butanedithiol, PEG dithiol, 1,6-hexanedithiol,2,2′-thiodiethanethiol, ethane-1,2-diyl bis(3-mercaptopropanoate),hexa(ethylene glycol) dithiol, tetra(ethylene glycol) dithiol,1,16-hexadecanedithiol, benzene-1,2-dithiol, benzene-1,3-dithiol,benzene-1,4-dithiol, biphenyl-4,4′-dithiol, p-terphenyl-4,4″-dithiol,(S)-2-aminobutane-1,4-dithiol hydrochloride,4-phenyl-4H-(1,2,4)triazole-3,5-dithiol,5-(4-chlorophenyl)-pyrimidine-4,6-dithiol, 1,4-benzenedimethanethiol,2-mercaptoethyl ether, or L-(−)-dithiothreitol.
 3. The compositematerial of claim 1, wherein the polymer is derived from the firstmonomer, the first cross-linker, and a second monomer, wherein thesecond monomer comprises two terminal carbon-carbon double bonds.
 4. Thecomposite material of claim 3, wherein the second monomer istri(ethylene glycol) divinyl ether (TEGDV), 1,9-decadiene,1,4-bis(vinyloxy)butane, diallylphthalate, diallyl diglycol carbonate,poly(ethylene glycol) divinyl ether, divinyl glycol, divinylbenzene,divinyl sulfone, 1,4-butanediol divinyl ether, allyl ether, allylsulfide, 1,4-bis(4-vinylphenoxy)butane, 1,5-hexadiene, dipentene,(R)-(+)-limonene, (S)-(−)-limonene, N,N′-methylenebis(acrylamide), orN,N′-ethylenebis(acrylamide).
 5. The composite material of claim 4,wherein the polymer is derived from the first monomer, the firstcross-linker, the second monomer, and a third monomer, wherein the thirdmonomer is (+)-N,N′-diallyltartramide (DATA), diallyl disulfide, diallylcarbonate, diallyl maleate, diallyl succinate, trimethylolpropanediallyl ether, 1,1-diallyl-1-docosanol, 1,1-diallyl-3-(1-naphthyl)urea,1,1-diallyl-3-(2-ethylphenyl)urea, 1,2-diallyl-1,2-cyclohexanediol,2,6-diallyl-meta-cresol, N,N-diallyl-2-hydroxypropanamide,1,4-pentadien-3-ol, trimethyl(propargyl)silane, or propargylamine. 6.The composite material of claim 1, wherein the first cross-linker is1,3,5-triallyl-1,3,5-triazine-2,4,6(1H,3H,5H)-trione (TATATO),1,6-heptadiyne, 1,7-octadiyne, 1,8-nonadiyne, 1,9-decadiyne, propargylacrylate, 4-arm PEG norbornene, trimethylolpropane triacrylate,tetra-alkyne poly(ethylene glycol), 2,4,6-triallyloxy-1,3,5-triazine,triallylamine, triallyl borate, triallylphosphine, diallyl fumarate,3-(allyloxy)-1-propyne, glyoxal bis(diallyl acetal), tetraallylsilane,propargyl ether, or squalene.
 7. The composite material of claim 1,wherein the cross-linked gel further comprises a plurality of graftedend-groups.
 8. The composite material of claim 7, wherein the graftedend-groups are derived from a molecule having a thiol functional groupor a molecule having an unsaturated carbon-carbon bond.
 9. The compositematerial of claim 8, wherein the grafted end-groups are derived from amolecule having a thiol functional group; and the molecule having athiol functional group is 3-mercaptopropionic acid, 1-mercaptosuccinicacid, a peptide having a cysteine residue, a protein having a cysteineresidue, cysteamine, 1-thiohexitol, poly(ethylene glycol)2-mercaptoethyl ether acetic acid, poly(ethylene glycol) methyl etherthiol, 1-thioglycerol, 2-naphthalenethiol, biphenyl-4-thiol,3-amino-1,2,4-triazole-5-thiol, 5-(trifluoromethyl)pyridine-2-thiol,1-[2-(dimethylamino)ethyl]-1H-tetrazole-5-thiol, 1-propanethiol,1-butanethiol, 1-pentanethiol, 1-hexanethiol, 1-octanethiol,8-amino-1-octanethiol hydrochloride,3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluoro-1-octanethiol,8-mercapto-1-octanol, or γ-Glu-Cys.
 10. The composite material of claim7, further comprising a first ligand, wherein the first ligandcomprises: an ion exchange functionality, a hydrophobic interactionmoiety, or a biomolecule; and at least one grafting group.
 11. Thecomposite material of claim 10, wherein the grafting group is anunsaturated carbon-carbon bond.
 12. The composite material of claim 10,wherein the grafting group is a thiol functional group.
 13. Thecomposite material of claim 10, wherein the first ligand is abiomolecule comprising at least one thiol functional group.
 14. Thecomposite material of claim 13, wherein the biomolecule is a protein Aderivative.
 15. The composite material of claim 10, further comprising asecond ligand, wherein: the polymer is derived from the first monomer,the first cross-linker, and a second monomer; the first ligand is abiomolecule comprising a least three thiol functional groups; the secondmonomer comprises two terminal carbon-carbon double bonds; the secondligand is a biomolecule comprising at least three thiol functionalgroups; and the second ligand is a second cross-linker.
 16. Thecomposite material of claim 15, wherein the second ligand is a protein Aderivative.
 17. The composite material of claim 15, wherein the secondmonomer is poly(ethylene glycol) divinyl ether.
 18. The compositematerial of claim 15, further comprising a third ligand, wherein: thepolymer is derived from the first monomer, the first cross-linker, thesecond monomer, and a third monomer; the third monomer comprises twoterminal carbon-carbon double bonds; the third ligand is a biomoleculecomprising at least three thiol functional groups; and the third ligandis a third cross-linker.
 19. The composite material of claim 18, whereinthe third ligand is a protein A derivative.
 20. The composite materialof claim 18, wherein the third monomer is poly(ethylene glycol) divinylether.
 21. The composite material of claim 1, wherein the compositematerial is a membrane.
 22. A method, comprising the step of: contactingat a first flow rate a first fluid comprising a substance with acomposite material of claim 1, thereby adsorbing or absorbing a portionof the substance onto the composite material.